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Article

Solid Microbial Fertilizers Prepared with Different Carriers Have the Potential to Enhance Plant Growth

by
Lianhao Sun
1,
Yuexiang Zhou
1,
Hui Nie
1,
Chong Li
1,2,
Xin Liu
1,
Jie Lin
1,
Xiongfei Zhang
3 and
Jinchi Zhang
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Jiangsu Province Key Laboratory of Soil and Water Conservation and Ecological Restoration, Nanjing Forestry University, Nanjing 210037, China
2
Department of Renewable Resources, University of Alberta, Edmonton, AB T6G 2E3, Canada
3
Jiangsu Academy of Forestry, Nanjing 211153, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(3), 539; https://doi.org/10.3390/f16030539
Submission received: 12 February 2025 / Revised: 9 March 2025 / Accepted: 18 March 2025 / Published: 19 March 2025
(This article belongs to the Section Forest Soil)
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Abstract

:
Microbial inoculants are vital for promoting plant growth and facilitating the ecological restoration of degraded forested regions near abandoned mine sites. However, the direct application of liquid microbial inoculants is often challenging due to low microbial activities and poor transport efficiencies, which limit their effectiveness in complex soil environments. To tackle these challenges, this study utilized immobilized microbial technology to evaluate the effectiveness of solid microbial inoculants sourced from peat (P), biochar (BC), and spent mushroom substrates (SMSs) in enhancing the soil’s multifunctionality and promoting plant growth. Specifically, this research sought to assess the effectiveness of solid microbial inoculants derived from peat (P), biochar (B), and spent mushroom substrates (SMSs) in enhancing soil multifunctionality and promoting plant growth in nutrient-deficient soils that were affected by abandoned mine sites. We aimed to evaluate the performance of different solid microbial inoculants in improving the soil’s nutrient content and enzyme activities. A 24-week pot experiment was conducted using Medicago sativa L. in nutrient-poor soil. The results demonstrated that, in contrast to peat and biochar, SMSs effectively interacted with microbial inoculants and significantly improved the nutrient content and enzyme activities of nutrient-deficient soil. It was noted that β-1,4-glucosidase (BG), invertase, β-1,4-N-acetylglucosaminidase (NAG), urease, and soil available phosphorus increased by 204%, 405%, 118%, 198%, and 297%, respectively. The soil’s multifunctionality improved by 320% compared with the CK, and the plant biomass also increased significantly. Further, our random forest analysis indicated that the soil available phosphorus, ammonium nitrogen, total nitrogen, total carbon content, arylsulfatase, pH, total phosphorus, NAG, and BG were key environmental factors that induced changes in plant biomass. These findings highlighted the potential of SMSs as an effective carrier for immobilized microbial inoculants, which provides a sustainable approach for the restoration of forest soils surrounding abandoned mine sites, as well as a promising avenue for the valorization of agricultural waste.

1. Introduction

The rapid advancement of the global economy has resulted in a substantial increase in the demand for mineral resources [1]. The rapid growth of the mining industry has inevitably caused damage to original vegetation ecosystems on slopes, as well as soil erosion, resulting in numerous rocky slopes and exposed hillside areas, which have led to the further continuous degradation of forested mining areas [2]. Consequently, the restoration and reconstruction of the natural ecological vegetation of damaged slopes to alleviate forest land degradation has become particularly urgent. The establishment of vegetation cover to stabilize mining slopes is widely regarded as an ideal long-term solution, as it is an environmentally compatible, sustainable, and relatively economical technology. However, the long-term stable growth of plants is often compromised due to unsuitable physical and chemical conditions for their growth in abandoned mining areas, which encompass high levels of toxic metals, extremely high acidity, low nutrient contents, as well as poor soil matrix structures and water retention capacities [3]. Recent studies have shown that microorganisms play crucial regulatory roles in the bioremediation of degraded soils [4]. Certain functional microorganisms, such as plant-growth-promoting rhizobacteria (PGPR), perform essential roles in nutrient acquisition and assimilation. Furthermore, they enhance the soil structure and secrete/regulate extracellular molecules (e.g., hormones, secondary metabolites, antibiotics, and various signaling compounds), thereby improving the physical and chemical properties of soils while stimulating plant growth and development [5]. Our prior investigation involved the assessment of various functional microorganisms that demonstrated effective capabilities in reducing the soil’s pH, facilitating the liberation of calcium and magnesium ions, contributing to soil formation, enhancing nutrient availability, and fostering plant growth [6,7]. The strain Bacillus thuringiensis NL-11, obtained from soil in the vicinity of weathered dolostones, has demonstrated potential through the secretion of organic acids that facilitate rock weathering, thereby assisting plants in developing robust root systems. However, when functional microorganisms are directly applied to the soil, they often encounter issues with reduced activities and poor stability [8]. Concurrently, their functions may be hindered by competition from indigenous microorganisms or by the soil’s physical and chemical properties, which diminish their performance and adversely affect the efficacy of ecological restoration [9]. To tackle this issue, advancements in bioinput formulations are being made to address challenges related to stability, viability, shelf life, and application methods. Furthermore, the creation of alternative bioinput delivery systems continues to be a priority within efforts designed to improve the efficacy of bioproducts in field applications.
Microbial immobilization technology enhances the tolerance, activities, and stability of selected functional microorganisms by immobilizing them onto specific carriers, thereby facilitating the preservation of microbial inoculants [10]. This technology provides a conducive microenvironment for microorganisms, reduces competition between exotic and native microbes, and assists with overcoming poor soil conditions, thereby promoting the survival and colonization of beneficial microorganisms [11]. These carriers, being a crucial element of inoculant formulations, must exhibit favorable physical and chemical properties, be easy to produce, be safe to handle, have no detrimental environmental impacts, and possess an extended shelf life [12]. Peat is among the most widely utilized organic carriers for rhizobia across North America, South America, Europe, and Australia and has served as a benchmark for assessing new carriers [13]. Nevertheless, peat is limited and costly in many regions of Asia and Africa [14]. Moreover, the extraction of peat is giving rise to growing environmental concerns. Biochar is regarded as an effective organic carrier owing to its neutral pH, high bulk density, strong adhesion, low toxicity, and minimal contamination issues [15]. However, the production, transport, and application costs associated with biochar remain the major factors that limit its widespread use. Spent mushroom substrates (SMSs) represent a byproduct of mushroom cultivation [16]. Increased mushroom production has led to the significant generation of SMSs [17]. In certain countries, the absence of SMS waste management poses environmental hazards. Consequently, the recycling and reuse of SMSs would be both cost-effective and environmentally sustainable. As a carrier, SMSs possess a loose texture and advantageous pore structure that promotes the attachment and growth of microorganisms [18]. When combined with functional microorganisms, the distinctive structure of SMSs can enhance the survival and colonization potential of these microorganisms in the soil [19]. Moreover, SMSs offer a nutrient-rich environment that supports microbial activities and promotes the growth of microbial strains, consequently enhancing the nutritional status and microbial diversity of rhizospheric soil [20]. This creates new research avenues and opportunities for the application of functional microorganisms in the ecological restoration of abandoned mine sites.
Soil plays a pivotal role in ecological restoration technologies for degraded forests in proximity to abandoned mines, as it serves as the primary medium for supplying water and essential nutrients to plants [21]. Furthermore, soil concurrently sustains numerous ecosystem functions and services—a concept known as soil multifunctionality—which is generally managed or assessed based on a variety of soil functions or processes that are vital for sustaining productivity [22]. There is a strong correlation between soil multifunctionality and plant biomass, which renders it essential for nutrient cycling. Although the beneficial effects of functional microorganisms on soil’s nutrient levels are well documented, the influence of solid microbial agents, which are developed with diverse carriers, on the comprehensive multifunctionality of soil—such as the cycling of carbon, nitrogen, phosphorus, and sulfur—has yet to be thoroughly investigated [23]. In response to this issue, we conducted a pot experiment utilizing peat, biochar, and SMSs as carriers, while selecting Bacillus thuringiensis NL-11 as the microbial inoculant, given that this strain has exhibited plant-growth-promoting effects in previous research. This experiment comprised eight treatments with five replicates and was conducted over a period of six months, during which quantitative analyses were performed to evaluate soil nutrients, enzyme activities, and other indicators, which led to the establishment of a soil multifunctionality index. Our objective was to explore the following hypothetical questions: (1) In what ways do various carriers influence the efficacy of microbial inoculants in enhancing soil’s nutrient availability and enzyme activities? (2) What are the overall effects of these carriers and inoculants on soil multifunctionality and plant growth in the degraded forest soils surrounding mine sites? By leveraging the synergistic effects of carriers and functional microorganisms, we sought to enhance plant growth and promote long-term microbial colonization on degraded forest slopes. Moreover, this research provides theoretical perspectives and practical methodologies for ecological restoration at deserted mine sites.

2. Materials and Methods

2.1. Carriers and Strains

For this experiment, we chose peat (P), biochar (BC), and spent mushroom substrates (SMSs) as microbial carriers. The detailed material descriptions are as follows: The peat was acquired in Lithuania. The biochar was generated by pyrolyzing wood waste at a high temperature of 600 °C. The spent mushroom substrates utilized in this experiment were sourced from Pleurotus ostreatus [18]. During the preparation of the carriers, the three materials under study were air-dried at room temperature for three to five days and subsequently crushed after complete drying. The materials were then sieved through a 2 mm mesh to ensure uniformity and a suitable particle size of the carriers. The fundamental physicochemical properties of the three carriers are presented in Table S1.
B. thuringiensis NL-11 was isolated from soil surrounding weathered dolomite [7]. In this experiment, B. thuringiensis NL-11 was inoculated into a liquid culture medium and fermented in a shaking flask at 30 °C and 200 r/min for a duration of 48 h. The microbial suspension was then diluted to 108 colony-forming units (CFU) mL−1 using sterile distilled water [24]. The cell concentration was determined based on optical density measurements.

2.2. Laboratory Preparation of Immobilized Microbial Inoculant and Assessment of Shelf Life

To prepare the immobilized microbial agents, 100 g of air-dried carrier samples underwent two sterilization cycles in an autoclave, maintained at 121 °C for 15 min each. Following sterilization, the samples were placed in a clean bench to cool to room temperature and subsequently transferred to polyethylene bags. Following this, a suitable volume of sterile water was incorporated, and the carrier was thoroughly mixed with the water to achieve uniform moisture distribution. The liquid microbial inoculant was then added to the bags at a ratio of 80% (v/w, bacterial inoculant/carrier), mixed thoroughly, and sealed. To promote oxygen circulation, six small puncture holes were randomly created in each bag, thereby allowing for gas exchange. Upon completion of these procedures, the bags containing the liquid microbial inoculant were placed in an incubator set at 28 °C for cultivation.

2.3. Design of Experiments and Sample Collection

The experiment was carried out in March 2022 at the Xiashu Forestry Station greenhouse (31°7′ N, 119°12′ E) and lasted for a duration of 6 months. The temperature in the greenhouse was maintained at between 18 °C and 35 °C, with a relative humidity that ranged from 40% to 80%. The duration of the photoperiod fluctuated between 10 and 14 h, accompanied by a midday photosynthetic photon flux density (PPFD) of approximately 1000 μmol/m2/s. Topsoil, ranging from 0 to 30 cm in depth and sourced from the station, served as the substrate, which is frequently employed for the rehabilitation of adjacent abandoned mining locations. Based on the classification system outlined by the United States Department of Agriculture (USDA) Soil Taxonomy, this soil was identified as Ultisol, which is characterized by its loamy clay texture. Following a period of air-drying lasting 3 to 5 days, the soil was subjected to processing through a 5 mm sieve to achieve uniformity. The nutrient characteristics of the soil, as detailed in Table S2, were as follows: pH, 5.31; total carbon content, 0.04%; total sulfur content, 0.034%; total nitrogen content, 0.51%; total phosphorus content, 1.23 g/kg; available phosphorus, 2.1 mg/kg; and soil organic carbon content, 0.33%. A total of eight treatment groups were created, which included (CK) inactivated liquid microbial inoculant; (B. thuringiensis NL-11) activated liquid microbial inoculant; (P-) 100 g of peat substrate; (P) 100 g of peat microbial inoculant; (BC-) 100 g of biochar; (BC) 100 g of biochar microbial inoculant; (SMS-) 100 g of spent mushroom substrates; and (SMS) 100 g of spent mushroom substrates microbial inoculant, as summarized in Table S3. Medicago sativa L. was chosen as the pioneer species to improve the soil stability and facilitate the restoration of the mining site. Each pot (Ø30 cm at the top and 23.5 cm in height) was filled with 5 kg of soil, to which solid microbial fertilizer, consisting of one of the three chosen carriers, was incorporated and thoroughly blended. Prior to sowing, uniformly sized whole alfalfa seeds were immersed in warm water for 25 min to soften the seed coat, sterilized using a 10% H2O2 solution for 15 min, and subsequently rinsed three to five times with sterile water before air-drying and planting. The seeds were uniformly distributed, and after a period of two weeks, the seedlings were reduced to twenty plants. After six months of growth, destructive sampling was conducted, and ten intact plants were harvested from each pot to assess their fresh weight. Subsequently, the plants were dried in an oven at 75 ± 8 °C until a constant weight was attained to quantify the biomass of the samples. Soil samples were obtained from the rhizosphere of the plants utilizing a sterilized knife, which was disinfected with 75% ethanol following each sample collection. Each pot was utilized for plant extraction, and gentle shaking was applied to dislodge any loosely attached soil from the roots. The remaining soil adhering to the roots (approximately 1 mm in thickness) constituted the rhizosphere. The gathered soil samples were separated into two portions. One portion was air-dried in the laboratory to analyze the soil nutrients, while the other was stored in a refrigerator at 4 °C to assess the soil enzyme activities.

2.4. Assessment of Soil Nutrients and Enzymatic Activities

This research identified ten indicators of soil nutrients that are crucial for plant growth, which encompass total carbon in soil (TC), organic carbon in soil (SOC), total phosphorus (TP), available phosphorus in soil (AP), and total sulfur (TS). The soil pH was first assessed with a PB-10 pH meter (Sartorius GmbH, Göttingen, Germany) following an hour of shaking, utilizing a ratio of 1:2.5 for soil to deionized water [25]. Total nitrogen (TN), total carbon (TC), and total sulfur (TS) were quantified with an elemental analyzer (Vario MAX cube; Elementar, Germany). Ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) were extracted by mixing the soil with 2 M KCl in a 1:5 ratio and agitating at 250 rpm for 60 min at 25 °C for subsequent analysis with a spectrophotometer (UV 2700, Shimadzu, Japan) (LY/T1230-1999 [26] and LY/T1231-1999 [27]). Available phosphorus was extracted with a 0.5 M sodium bicarbonate solution and quantified using the methylene blue colorimetric method (LY/T1230-1999). Available potassium was extracted with a 1 M ammonium acetate solution and quantified with a flame photometer (LY/T1236-1999 [28]), while total potassium (TK) was assessed using the same device (LY/T1234-1999 [29]).
In this research, we assessed six extracellular soil enzymes that play a role in elemental cycling, specifically C-cycle enzymes (β-1,4-glucosidase and invertase), N-cycle enzymes (β-1,4-N-acetylglucosaminidase and urease), P-cycle enzymes (acid phosphatase), and S-cycle enzymes (arylsulfatase). The activities of β-1,4-glucosidase were quantified using p-nitrophenyl-β-D-glucopyranoside as a substrate, whereas invertase activities were assessed via the 3,5-dinitrosalicylic acid (DNS) method [30]. Further, the activities of β-1,4-N-acetylglucosaminidase were determined using an improved p-nitrophenyl release colorimetric method, and those of urease were quantified using urea as a substrate through steam distillation [31]. Phosphatase activities were evaluated with disodium phenyl phosphate as a substrate, whereas arylsulfatase activities were determined by quantifying the release of p-nitrophenol from potassium p-nitrophenyl sulfate [32].

2.5. The Multifunctionality of Soil and the Roles of Carbon, Nitrogen, Phosphorus, and Sulfur Cycles

We subsequently evaluated the multifunctionality of soil through the quantification of 14 distinct functions, which encompassed the following key areas: (a) carbon cycling within the soil, which includes total carbon (TC), soil organic carbon (SOC), β-glucosidase (BG), and invertase (INV); (b) nitrogen cycling in the soil, which measures total nitrogen (TN), nitrate nitrogen (NO3−-N), ammonium nitrogen (NH4+-N), β-1,4-N-acetyl-glucosaminidase (NAG), and urease (URE); (c) pertinent indicators of phosphorus cycling in the soil, such as acid phosphatase and effective phosphorus; (d) sulfur cycling in the soil, which involves determining the total sulfur (TS) and arylsulfatase (AS) [31]. These functions are closely interconnected with the cycling and storage of carbon, nitrogen, phosphorus, and sulfur within the soil, thus indicating its ecological service potential [33]. To calculate the soil multifunctionality, an averaging method was employed to analyze the aforementioned functions. Initially, each individual function in the soil was standardized and normalized using z-score transformation, after which the standardized data were utilized to calculate the average of the 14 soil functions [21].

2.6. Statistical Analysis

The data were subjected to statistical evaluation utilizing GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). The Shapiro–Wilk test was employed to evaluate the normality of variance regarding the soil’s chemical properties and plant biomass data. For data that followed a normal distribution with homogeneous variance, one-way ANOVA and turkey post hoc were employed for multiple comparisons. The Kruskal–Wallis test alongside Dunn’s post hoc analysis were utilized for multiple comparisons involving non-Gaussian or heteroscedastic data. The bar chart and scatter plot were created utilizing GraphPad Prism 9.0. Random forest regression was employed to identify keystone microbial taxa that impacted the plant biomass, utilizing the “random Forest” package in R. The relationship between environmental factors and plant biomass was assessed using the Kruskal–Wallis and spearman correlations with the “Lin KET” package in R.

3. Results

3.1. Responses of Alfalfa Biomass to Different Carriers Mediated by Microbial Inoculants

In the pot experiment after six months of cultivation, significant growth differences were observed for the alfalfa between the different treatment groups (Figure 1a,b) (p < 0.05). In comparison to the CK and NL-11 groups, those employing immobilized microbial inoculants with various carriers (e.g., P+ group, BC+ group, and SMS+ group) demonstrated significantly more robust alfalfa growth. All these treatments increased the fresh and dry weights of alfalfa to varying degrees. Notably, the SMS+ treatment group demonstrated the most favorable results, significantly surpassing those of the CK, NL-11, and SMS- groups. These findings indicated that the application of immobilized microbial inoculants exerted a significantly positive influence on the alfalfa biomass.

3.2. Responses of Rhizospheric Physicochemical Properties of Soil and Enzyme Activities Associated with Alfalfa to Different Carriers Mediated by Microbial Inoculants

The addition of microbial inoculants using different carriers exhibited significant differences in the carbon, nitrogen, and phosphorus contents in the soil. In comparison to the CK group, the physicochemical characteristics of the rhizospheric soil associated with alfalfa exhibited enhancements across all inoculation treatment groups. Notably, for the SMS+ group, the organic carbon, nitrate nitrogen, ammonium nitrogen, and available phosphorus contents of the rhizospheric soil increased by 37.3%, 25%, 36%, and 23%, respectively, and exhibited the best improvement effect between treatments (Figure S1) (p < 0.05). Additionally, the integration of SMSs with B. thuringiensis NL-11 resulted in a moderate increase in the soil pH, consequently postponing soil acidification and fostering robust plant growth (Figure S1i).
The impact of the synergistic application of microbial inoculants combined with various carriers on enzyme activities within the rhizospheric soil of plants is depicted in Figure S2. Compared to the CK group, enzyme activities in the rhizospheric soil of alfalfa treated with different carrier matrices were generally increased. Among the activities of BG, INV, NAG, URE, and AS, the enhancement observed in the SMS+ group was the most significant across all treatment groups (p < 0.05). In comparison to the CK group, the activities of these enzymes increased by 204%, 405%, 118%, 198%, and 297%, respectively. Notably, although the application of microbial inoculants or SMSs alone also resulted in a moderate increase in enzyme activities within the rhizospheric soil of alfalfa, the enhancement was less pronounced than that observed in the SMS+ group. These findings suggest that the synergistic interactions between microbial inoculants and carriers significantly enhanced the physicochemical properties and enzyme activities in the rhizospheric soil of alfalfa, thereby facilitating plant growth and development, while also contributing to the enhancement of soil health and ecological functions.

3.3. Responses of Multifunctionality in Rhizospheric Soil of Alfalfa to Different Carriers Mediated by Microbial Inoculants

Under various carrier treatments, a Pearson correlation analysis was conducted to evaluate the relationship between alfalfa biomass and soil nutrients, as well as enzyme activities. A significantly positive correlation (p < 0.05) was observed between the alfalfa fresh weight and biomass with soil TC, TN, NH4+-N, TP, and AP (Figure 2). Further random forest analysis indicated that SOC, AP, TN, NH4+-N, TC, AS, pH, TP, NAG, and BG had impacts on the growth of alfalfa, while URE, ACP, NO3−-N, INV, and TS did not affect plant growth (p < 0.05) (Figure 3). These findings indicate a strong relationship between the increase in alfalfa biomass and the enhancement of soil nutrients and enzyme activities, suggesting that the combined application of microbial inoculants and various carriers improved the nutrient status and biological activities of the soil, thereby promoting plant growth and development. To enable a comprehensive comparison of the impacts of microbial inoculation alongside various carrier treatments on soil nutrient levels and enzyme activities, we combined these parameters into a soil multifunctionality index. The findings indicated that the incorporation of microbial inoculants substantially improved the multifunctionality of soil within the groups that were subjected to carrier treatments. Notably, in the SMS treatment group, the soil multifunctionality increased by 320% (p < 0.05) (Figure 4). Further, the soil multifunctionality index demonstrated a positive correlation with multiple soil nutrient and enzyme activity indicators, as well as a significantly positive correlation with the fresh and dry weights (p < 0.05). The analytical findings demonstrated that the solid microbial inoculant treatment employing SMSs as a carrier exhibited the most pronounced enhancement of soil multifunctionality. These findings suggested that the application of microbial inoculants, particularly in conjunction with suitable carriers, can significantly enhance the multifunctionality of soil, thereby contributing to its improved ecological services (Figure 4). In summary, a well-considered strategy for the application of microbial inoculants has the potential to effectively promote plant growth and improve soil health, thereby showcasing considerable application value for the ecological rehabilitation of abandoned mining sites.

4. Discussion

We administered B. thuringiensis NL-11, a microbial inoculant that facilitates mineral solubilization, to enhance rock decomposition and nutrient liberation, thereby promoting root growth and plant viability. Our investigation indicated that immobilized microbial inoculants with various carriers had a significant impact on plants, with the SMS carrier demonstrating the most pronounced stimulating effect. This enhancement may be primarily attributed to two causes. Firstly, the solid microbial inoculants that were prepared jointly with SMSs and B. thuringiensis NL-11 not only contained a rich supply of organic matter, nitrogen, phosphorus, and potassium, among other macronutrients, but also included small molecularly active substances secreted by microorganisms [34]. These components can effectively promote plant metabolism, as well as stimulate crop growth and development [35]. Secondly, functional microorganisms can colonize the root systems of host plants to form beneficial symbiotic relationships. On the one hand, they convert chemical substances in the soil into readily absorbable nutrients and small molecularly active substances for the plants [36]. On the other hand, these microorganisms improve the microbial ecological environment of the soil, which further promotes healthy plant development [37]. Research has indicated that microbial inoculations can stimulate Aspergillus communities in the soil to thereby synergistically promote plant growth [38].
SMSs are rich in various nutrients that can enhance soil’s quality, promote microbial activities, and improve plant health [18]. The introduction of SMSs into the soil leads to a cascade of biochemical transformations. This includes a rise in the diversity of bacterial and fungal populations, the enrichment of particular microorganisms, the synthesis of antioxidant enzymes, and the improvement of metabolic pathways [39]. Moreover, the combination of microbial inoculants and SMSs can significantly enhance nitrogen accumulation in soil, lessen the cycling of nitrogen in ecosystems, and address soil acidification challenges in areas that are affected by mining activities [40]. This experiment indicated that immobilized carriers enhanced the effectiveness of microbial inoculants [41]. However, as “invaders”, microbial inoculants may compete with local microbe species for resources, which potentially impacts their chances of survival, ultimately leading to the variable efficacy of their invasion [42]. Although carriers contribute a significant quantity of organic matter to the soil, they concurrently safeguard the beneficial microorganisms that adhere to their surfaces [43]. The application of SMSs enhances the activities of functional microorganisms in the soil, thereby assuring their dominance in the soil environment [44]. The results of this study demonstrated that SMSs served as an equally effective carrier for inoculants in comparison to peat and biochar. Therefore, especially for the many countries where peat is difficult to obtain, SMSs can serve as an alternative carrier. Further, compared with the economic costs of the production, transport, and application of biochar, the high water retention and high porosity of SMSs as a carrier can diminish the requirements for temperature and humidity during transportation. Thus, SMSs (as an agricultural waste product) are both economical and environmentally compatible.
In this investigation, the soil pH level in the SMS treatment group exceeded that of the CK group, suggesting that SMSs possess the ability to regulate soil’s pH [20,45]. This phenomenon may be ascribed to the organic acid anions generated from the decomposition of organic matter linked to H+, resulting in an increase in the soil pH [46]. This was consistent with earlier findings, where the inoculation of Pseudomonas H1 significantly elevated the soil pH of soybeans, decelerated soil acidification, and promoted soybean growth [24,47]. The combination of microbial inoculants with SMSs exhibited the most significant effects for increasing the soil pH compared with the application of SMSs alone. This may be attributed to the addition of SMSs, which enhanced soil aggregation, porosity, nutrient status, and enzyme activities, thus creating a suitable environment for the colonization of B. thuringiensis NL-11 in the soil [48]. Furthermore, the application of SMSs significantly elevated the organic carbon content in the soil compared to peat and biochar, while facilitating strain growth and enhancing nutrient release, thereby further augmenting the soil’s organic carbon content [49]. Previous studies also indicated that applied inoculants contained various beneficial microorganisms that increased the availability of soil nutrients, thereby promoting plant growth [50].
Soil enzymes play a crucial role in the transference of organic matter and nutrient cycling, rendering them significant indicators of soil ecosystem health and sustainability [51]. Previous studies have reported a significant enhancement in soil enzyme activities following the introduction of functional microorganisms. In this study, the B. thuringiensis NL-11 and SMS treatments significantly enhanced the soil enzyme activities compared with the CK soil [52,53]. Elevated soil enzyme activities following the inoculation of PGPR strains have been previously reported. For instance, the activities of soil enzymes were significantly improved following the inoculation of Bacillus subtilis in comparison to the uninoculated CK group [54]. The increased soil enzyme activities associated with SMS amendments corresponded with the results obtained from various organic matter improvement cultivation practices [55]. The decomposition of SMSs may improve the availability of nutrients (C, N, P) in the soil [56], which is advantageous for the growth of indigenous microorganisms and stimulates soil enzyme activities [57]. The application of PGPR in conjunction with SMSs led to the observation of the most elevated soil enzyme activities [58]. Likewise, earlier research demonstrated that the combined use of PGPR and biochar improved soil’s sucrase activities [59]. Previous research also reported on the maximum activities of urease, dehydrogenase, and phosphatase in treatments that involved waxy Bacillus and biochar [60,61]. Consequently, the combined application of B. thuringiensis NL-11 and SMSs was most advantageous for stimulating soil nutrient cycling, and served as an important factor for enhancing plant growth.
Soil multifunctionality represents the cumulative value of various ecological service functions and processes [62], which varies with each functional performance and is influenced by a range of factors [63,64]. Consequently, we utilized a soil multifunctionality index to thoroughly evaluate the soil’s nutrients and enzyme activities to identify a significantly positive correlation among soil nutrients [65], enzyme activities, and plant growth (p < 0.05). This suggested that alterations in soil multifunctionality, facilitated by microbial inoculants and carriers, were a primary driver of changes in plant growth [2]. This finding supported previous research, which demonstrated that fertilization measures [66], particularly organic amendments, positively impacted soil multifunctionality by promoting regulatory and supportive services related to soil quality and the protection of biodiversity [67]. The potential mechanisms for enhancing the soil multifunctionality likely involved several aspects [68]. First, the application of SMSs elevated the soil pH from 5.54 to 6.32, with the soil pH serving as a crucial abiotic factor that significantly influenced the resilience and recovery of soil multifunctionality, potentially affecting it by modifying the soil’s microbial community structures [58,69]. Second, the increase in nutrients under SMS treatments might drive the maintenance of soil multifunctionality, as the application of immobilized microbial inoculants improves the availability of soil resources and stimulates the metabolism and composition of organic matter [70]. Generally, high soil multifunctionality is associated with elevated soil nutrient values. Ultimately, the introduction of immobilized microbial inoculants enhances soil enzyme activities, which serve as an early indicator of shifts in soil quality, exhibiting a markedly positive correlation with soil multifunctionality [71]. Therefore, the solid microbial inoculants prepared from SMSs and functional microorganisms not only maintained the multifunctionality of abandoned mine site soils but also significantly increased the biomass of alfalfa, which was critical for soil bioremediation under multifactorial stresses.

5. Conclusions

In summary, our experiment indicated that utilizing SMSs as a solid carrier for the fixation and adsorption of functional microorganisms significantly increased the nutrient content, enzyme activities, and multifunctionality of degraded soils of abandoned mine sites, thus enhancing the overall quality of the soil. Additionally, this synergistic effect significantly increased the biomass of both above-ground and below-ground components of alfalfa. Additionally, compared with the sole application of either functional microorganisms or SMSs, the combined application of both yielded the greatest improvements in soil multifunctionality and plant biomass. These findings indicated that the solid microbial inoculants derived from functional microorganisms and SMSs played a crucial role in the enhancement and restoration of degraded abandoned mine site soils. This research provides novel strategies for the restoration of degraded forest lands in proximity to abandoned mines and vegetation greening on a global scale, advancing ecological restoration and overall environmental improvements.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f16030539/s1; Table S1. Physicochemical properties of peat, biochar, and spent mushroom substrates as carrier materials; Table S2. Physicochemical properties of soil; Table S3. Experimental design; Figure S1. Effects of solid microbial agents prepared with different carriers on soil nutrients. Different letters indicate significant differences for each treatment (Duncan’s test, p < 0.05). CK: inactivated liquid microbial inoculant; NL11 active liquid microbial inoculant; treatments with peat substrate (P-: 100 grams of peat substrate, P: 100 grams of peat microbial inoculant); biochar treatments (BC-: 100 grams of biochar, BC: 100 grams of biochar microbial inoculant); and waste mushroom substrate treatments (SMS-: 100 grams of waste mushroom substrate, SMS: 100 grams of waste mushroom substrate microbial inoculant). TC: total carbon; TN: total nitrogen; TS: soil total sulfur; TP: total phosphorus; AP: available phosphorus; NH4+-N: ammonium N; NO3−-N: nitrate N; pH; SOC: soil organic carbon; Figure S2. Effects of solid microbial agents prepared with different carriers on soil enzyme activities. Different letters indicate significant differences for each treatment (Duncan’s test, p < 0.05). CK: inactivated liquid microbial inoculant); NL11: active liquid microbial inoculant; treatments with peat substrate (P-: 100 grams of peat substrate, P: 100 grams of peat microbial inoculant); biochar treatments (BC-: 100 grams of biochar, BC: 100 grams of biochar microbial inoculant); and waste mushroom substrate treatments (SMS-: 100 grams of waste mushroom substrate, SMS: 100 grams of waste mushroom substrate microbial inoculant). URE: urease; INV: invertase; ACP: acid phosphatase; AS: aryl sulfatase; NAG: β-1,4-N-acetyl-glucosaminidase; BG: β-1,4-glucosidase.

Author Contributions

L.S., H.N., C.L., and J.Z. designed the study. L.S., Y.Z., H.N., and X.L. collected the data. L.S., Y.Z., and X.L. analyzed the data. L.S. drafted the manuscript. L.S., H.N., J.L., X.Z., and C.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Jinchi Zhang extends their sincere gratitude for institutional financial endorsement received through multiple provincial-level research initiatives, including (1) The Jiangsu Provincial Science and Technology Plan (Project Identification Number BE2022420); (2) The Jiangsu Forestry Sector’s Innovative Technology Advancement Program (LYKJ [2021] No. 30); (3) The Scientific Investigation Program of Baishan National Park (2021ZDLY01); and (4) The Jiangsu Higher Education System’s Priority Academic Disciplinary Development Scheme (PAPD).

Data Availability Statement

Experimental datasets generated through this investigation may be obtained through legitimate requests directed to the corresponding author, subject to restricted access protocols as required under privacy protection regulations.

Acknowledgments

Special thanks are extended to Frank Boehm of Lakehead University for his exceptional contributions in professional proofreading services, particularly through meticulous linguistic refinement of the present research manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of solid microbial agents formulated with various carriers on plant growth. (a) Fresh weight of plants; (b) dry weight of plants. Different letters denote significant differences for each treatment (Duncan’s test, p < 0.05). CK: inactivated liquid microbial inoculant; NL11: active liquid microbial inoculant; treatments utilizing peat substrate (P-: 100 g of peat substrate; P: 100 g of peat microbial inoculant); biochar treatments (BC-: 100 g of biochar; BC: 100 g of biochar microbial inoculant); and waste mushroom substrate treatments (SMS-: 100 g of waste mushroom substrate; SMS: 100 g of waste mushroom substrate microbial inoculant).
Figure 1. Effects of solid microbial agents formulated with various carriers on plant growth. (a) Fresh weight of plants; (b) dry weight of plants. Different letters denote significant differences for each treatment (Duncan’s test, p < 0.05). CK: inactivated liquid microbial inoculant; NL11: active liquid microbial inoculant; treatments utilizing peat substrate (P-: 100 g of peat substrate; P: 100 g of peat microbial inoculant); biochar treatments (BC-: 100 g of biochar; BC: 100 g of biochar microbial inoculant); and waste mushroom substrate treatments (SMS-: 100 g of waste mushroom substrate; SMS: 100 g of waste mushroom substrate microbial inoculant).
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Figure 2. Relationships between environmental factors and soil multifunctionality. Asterisks denote statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001). TC: total carbon; TN: total nitrogen; TS: total soil sulfur; TP: total phosphorus; AP: available phosphorus; NH4+-N: ammonium nitrogen; NO3−-N: nitrate nitrogen; URE: urease; SOC: soil organic carbon; INV: invertase; ACP: acid phosphatase; AS: aryl sulfatase; NAG: β-1,4-N-acetyl-glucosaminidase; BG: β-1,4-glucosidase.
Figure 2. Relationships between environmental factors and soil multifunctionality. Asterisks denote statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001). TC: total carbon; TN: total nitrogen; TS: total soil sulfur; TP: total phosphorus; AP: available phosphorus; NH4+-N: ammonium nitrogen; NO3−-N: nitrate nitrogen; URE: urease; SOC: soil organic carbon; INV: invertase; ACP: acid phosphatase; AS: aryl sulfatase; NAG: β-1,4-N-acetyl-glucosaminidase; BG: β-1,4-glucosidase.
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Figure 3. Mean predictor importance in the random forest (percentage increase in mean squared error) indicates that TC (total carbon), TN (total nitrogen), TS (soil total sulfur), TP (total phosphorus), AP (available phosphorus), NH4+-N (ammonium N), NO3−-N (nitrate N), URE (urease), SOC (soil organic carbon), INV (invertase), ACP (acid phosphatase), AS (aryl sulfatase), NAG (β-1,4-N-acetyl-glucosaminidase), and BG (β-1,4-glucosidase) are key environmental factors that drive plant biomass. The percentage increase in the MSE (mean squared error) of the variables was utilized to assess the importance of these predictors, where elevated MSE% values suggested more significant predictors. The significance levels of the predictors (p < 0.05) are represented in blue. Note: ns denotes no significant effect, * denotes p < 0.05, and ** denotes p < 0.01.
Figure 3. Mean predictor importance in the random forest (percentage increase in mean squared error) indicates that TC (total carbon), TN (total nitrogen), TS (soil total sulfur), TP (total phosphorus), AP (available phosphorus), NH4+-N (ammonium N), NO3−-N (nitrate N), URE (urease), SOC (soil organic carbon), INV (invertase), ACP (acid phosphatase), AS (aryl sulfatase), NAG (β-1,4-N-acetyl-glucosaminidase), and BG (β-1,4-glucosidase) are key environmental factors that drive plant biomass. The percentage increase in the MSE (mean squared error) of the variables was utilized to assess the importance of these predictors, where elevated MSE% values suggested more significant predictors. The significance levels of the predictors (p < 0.05) are represented in blue. Note: ns denotes no significant effect, * denotes p < 0.05, and ** denotes p < 0.01.
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Figure 4. (a) A heat map illustrating the variation in soil nutrients and enzyme activities across various treatments. (b) Soil multifunctionality indices across various treatments. (c,e) Correlation between soil multifunctionality and plant fresh weight, as well as plant biomass. (d) The degree to which changes in soil pH under various treatments affect the soil multifunctionality. Distinct letters denote significant differences among each treatment (Duncan’s test, p < 0.05).
Figure 4. (a) A heat map illustrating the variation in soil nutrients and enzyme activities across various treatments. (b) Soil multifunctionality indices across various treatments. (c,e) Correlation between soil multifunctionality and plant fresh weight, as well as plant biomass. (d) The degree to which changes in soil pH under various treatments affect the soil multifunctionality. Distinct letters denote significant differences among each treatment (Duncan’s test, p < 0.05).
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Sun, L.; Zhou, Y.; Nie, H.; Li, C.; Liu, X.; Lin, J.; Zhang, X.; Zhang, J. Solid Microbial Fertilizers Prepared with Different Carriers Have the Potential to Enhance Plant Growth. Forests 2025, 16, 539. https://doi.org/10.3390/f16030539

AMA Style

Sun L, Zhou Y, Nie H, Li C, Liu X, Lin J, Zhang X, Zhang J. Solid Microbial Fertilizers Prepared with Different Carriers Have the Potential to Enhance Plant Growth. Forests. 2025; 16(3):539. https://doi.org/10.3390/f16030539

Chicago/Turabian Style

Sun, Lianhao, Yuexiang Zhou, Hui Nie, Chong Li, Xin Liu, Jie Lin, Xiongfei Zhang, and Jinchi Zhang. 2025. "Solid Microbial Fertilizers Prepared with Different Carriers Have the Potential to Enhance Plant Growth" Forests 16, no. 3: 539. https://doi.org/10.3390/f16030539

APA Style

Sun, L., Zhou, Y., Nie, H., Li, C., Liu, X., Lin, J., Zhang, X., & Zhang, J. (2025). Solid Microbial Fertilizers Prepared with Different Carriers Have the Potential to Enhance Plant Growth. Forests, 16(3), 539. https://doi.org/10.3390/f16030539

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