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Article

From Isolation to Plant Growth Evaluation: Development of a Streptomyces-Based Bioinput Using Spent Yeast and Composting Leachate

by
Mateus Torres Nazari
1,*,
Aline Rubert
2,
Vera Analise Schommer
1,
Bruna Strieder Machado
1,
Camila Vancini
3,
Daniela Dal Castel Krein
4,
Valdecir Ferrari
5,
Helen Treichel
6,
Luciane Maria Colla
1,2,4 and
Jeferson Steffanello Piccin
1,2,4
1
Graduate Program in Civil and Environmental Engineering (PPGEng), University of Passo Fundo (UPF), Passo Fundo 99052-900, Brazil
2
Graduate Program in Food Science and Technology (PPGCTA), University of Passo Fundo (UPF), Passo Fundo 99052-900, Brazil
3
Graduate Program in Agromomy (PPGAgro), University of Passo Fundo (UPF), Passo Fundo 99052-900, Brazil
4
Graduate Program in Chemical Engineering (PPGEQ), Federal University of Santa Maria (UFSM), Santa Maria 97105-900, Brazil
5
Graduate Program in Mining, Metallurgical and Materials Engineering (PPGE3M), Federal University of Rio Grande do Sul (UFRGS), Porto Alegre 90130-120, Brazil
6
Graduate Program in Environmental Science and Technology (PPGCTA), Federal University of Fronteira Sul (UFFS), Erechim 99700-970, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(10), 556; https://doi.org/10.3390/fermentation11100556
Submission received: 21 August 2025 / Revised: 20 September 2025 / Accepted: 25 September 2025 / Published: 27 September 2025
(This article belongs to the Special Issue Dissecting Actinobacteria: From Ecology and Biotechnological Value to Synthetic Biology)
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Abstract

This study aimed to develop a sustainable bioinput using Streptomyces sp. BEI-18A cultivated in an alternative culture medium (ACM) formulated with winery spent yeast and composting leachate. Actinomycetes were initially isolated from grape waste composting piles and screened for agricultural potential in vitro. Streptomyces sp. BEI-18A was selected for further bioinput development based on its high siderophore production. The ACM formulation was optimized in three steps: (I) determining the optimal concentration of winery spent yeast through mixture design; (II) assessing the effect of composting leachate addition on microbial growth; and (III) establishing the final composition of ACM components. The optimized ACM consisted of 3 g/L spent yeast, 2 g/L sucrose, 1 g/L soybean extract, and 10% (v/v) composting leachate. Cultivation of Streptomyces sp. BEI-18A in this medium resulted in a bioinput containing 7.80 × 107 CFU/mL. Its agricultural potential was validated in pot experiments with wheat and soybean, where application of the bioinput promoted significant improvements in early plant growth, enhancing several phytometric parameters. The results highlight the feasibility of valorizing agro-industrial residues as low-cost substrates for microbial bioinput production. This approach represents a promising strategy to foster sustainability in agriculture while reducing environmental impacts.

1. Introduction

Beneficial microorganisms are widely recognized as sustainable tools to promote plant growth, improve nutrient availability, and mitigate biotic and abiotic stresses [1,2,3,4]. Nevertheless, the Brazilian market remains dominated by Bacillus and Trichoderma [5,6]. Streptomyces, the main genus of actinomycetes, holds significant potential for agricultural applications; however, no Streptomyces-based bioinputs are currently registered or commercialized in Brazil [5,6,7]. This represents a missed opportunity, as these organisms produce phytohormones, siderophores, enzymes, and antifungal metabolites that enhance plant growth and stress tolerance [7,8,9,10].
In this context, bioprospecting Streptomyces isolates from diverse habitats and environmental conditions is crucial for identifying strains with unique adaptive traits relevant to agricultural applications. Composting systems offer a promising environment for isolating agriculturally relevant actinomycetes [11,12]. During composting, organic residues undergo microbially driven decomposition under dynamic conditions, fostering diverse microbial communities [13,14]. Actinomycetes are key contributors to organic matter degradation, and the selective pressures within composting environments favor strains with unique metabolic traits [15,16]. Thus, composting piles represent not only an environmentally friendly waste recycling strategy but also a valuable source for microbial bioprospecting.
Despite the potential of microbial bioinputs, high production costs remain a major barrier to large-scale application [17]. Conventional culture media are expensive, limiting the commercial viability of microbial products. Agro-industrial wastes, therefore, provide a sustainable alternative [18,19]. The grape industry, for instance, generates substantial waste during the grape processing; after wine fermentation and clarification, a semi-solid material known as spent yeast is produced, which represents 2–6% of the total wine volume [20,21,22,23,24]. This by-product is rich in nitrogen, phenolics, and residual sugars, offering a nutrient-dense substrate for microbial growth [20,21]. Composting leachate represents another low-cost option, enriched with soluble organic and inorganic compounds [25,26,27]. Utilizing these residues not only reduces production costs but also aligns microbial biotechnology with the principles of the circular economy [28].
In this study, we combined Streptomyces bioprospecting with the valorization of grape-processing wastes (e.g., spent yeast) and composting leachate to develop a novel and cost-effective bioinput. The objectives were to isolate and characterize actinomycetes with plant growth-promoting traits and to cultivate a Streptomyces strain in an alternative culture media. This dual approach addresses agricultural challenges while promoting sustainable waste management and advancing circular bioeconomy practices.

2. Materials and Methods

Figure 1 provides an overview of the methodological steps performed in this study, from actinomycete isolation and screening to culture medium development and bioinput evaluation.

2.1. Isolation and Identification of Actinomycetes

The actinomycete isolation stage began with the collection of samples from the upper layer (<20 cm) of grape waste composting piles. The composting process was carried out by Beifiur LTDA, located in Garibaldi, RS, Brazil (Latitude: −29.22857850; Longitude: −51.51184172). In the laboratory, 10 g of compost were transferred to 250 mL Erlenmeyer flasks containing 90 mL of saline solution (0.85% NaCl, Neon, New York, NY, USA). This suspension was shaken on a rotary shaker (Solab, Tokyo, Japan, SL-222) at 150 rpm for 5 min, followed by thermal treatment at 50 °C for 30 min. Serial dilutions were subsequently prepared (up to 10−5), and 0.1 mL aliquots of each dilution were spread onto Petri dishes containing ISP-2 medium (International Streptomyces Project). The ISP-2 medium was composed of 4 g/L yeast extract (Himedia, Thane, India), 10 g/L malt extract (Kasvi, Karnal, India), 4 g/L dextrose (Dinâmica, Barcelona, Spain), and 20 g/L agar (Merck, Darmstadt, Germany), and was sterilized at 121 °C for 20 min. Following inoculation, the plates were incubated (Solab, SL-101) at 28 °C for 7 to 14 days. During incubation, colonies exhibiting typical actinomycete morphology were selected for isolation and purification.
Purified isolates were examined by Gram staining. Isolates confirmed as Gram-positive were further characterized by 16S rRNA gene analysis for taxonomic identification at the genus level. Genomic DNA was extracted according to the methodology described by Sambrook and Russell [29], and the extracted DNA was used as a template for PCR amplification of the 16S rRNA gene. The primers employed were pA (5′-AGAGTTTGATCTGGCTCAG-3′) and 1492r (5′-TACGGTACCTTGTTACGACTT-3′), as described by Granada et al. [30]. The resulting ~1500 bp amplicons were sequenced, and the sequences were compared against those in the GenBank database using the BLAST algorithm (https://www.ncbi.nlm.nih.gov/ (accessed on 14 November 2022)) [31].

2.2. Microbial Screening Based on Agricultural Potential

The selection of the actinomycete strain investigated in this study was based on evaluating the isolates for their in vitro plant growth-promoting potential. Two parameters were assessed: siderophore production and indolic compound production.
Siderophore production by the microbial isolates was determined using a method adapted from Schwyn and Neilands [32]. Bacterial isolates were inoculated into five-fold diluted King’s B broth (Merck) and incubated at 28 °C for 48 h with shaking at 150 rpm. Following incubation, cultures were centrifuged at 12,000 rpm for 5 min. The resulting supernatant was mixed with chromo azurol S (CAS) reagent at a 1:1 ratio. The reaction mixture was kept at room temperature in the dark for 30 min, and absorbance was measured at 630 nm using a spectrophotometer (SpectraMax M5e, Agilent Technologies, Santa Clara, CA, USA). King’s B broth without microbial inoculation was used as the control. Siderophore production was evaluated based on the colorimetric shift in the CAS dye, reflecting the microorganisms’ ability to chelate iron ions.
Indolic compound production was assessed using a method adapted from Glickmann and Dessaux [33]. Isolates were inoculated into King’s B broth supplemented with 0.5 g/L tryptophan and incubated at 28 °C for 48 h with shaking at 150 rpm. After incubation, cultures were centrifuged at 12,000 rpm for 5 min, and the supernatant was mixed with Salkowski’s reagent [33] at a 1:1 ratio. The mixture was incubated in the dark for 30 min, after which absorbance was measured at 520 nm using a spectrophotometer (SpectraMax M5e). Indolic compound production was confirmed and quantified against a standard curve prepared using known concentrations of indolebutyric acid.

2.3. Development of Alternative Culture Medium and Bioinput Production

The development of the alternative culture medium (ACM) was carried out in three stages:
(a)
Determination of ACM formulation (Step I): The first stage focused on identifying the optimal ACM formulation for the growth of the selected actinomycete using a mixture design. The components evaluated included spent yeast (SY), sucrose (SC), soybean extract (SE), and ammonium sulfate (AS). Each formulation contained 12 g/L (m/v) of these components dissolved in deionized water. The primary objective was to assess the feasibility of SY as a constituent of the ACM. SY, a semi-solid residue derived from the wine clarification process, was supplied by a company in the Serra Gaúcha region, Brazil. Prior to use, SY was centrifuged (Eppendorf, Hamburg, Germany, Centrifuge 5810) at 3500 rpm for 10 min to separate the solid fraction. The remaining components (SE, SC, and AS) were obtained from local suppliers.
(b)
Incorporation of composting leachate (CL) (Step II): Based on the optimal formulation identified in Step I, the second stage aimed to incorporate composting leachate (CL) into the ACM. This step sought to evaluate CL as an alternative to potable water in microbial cultivation while providing a sustainable disposal route for this effluent. CL was tested at concentrations of 0%, 2.5%, 5%, 10%, 25%, 50%, and 100% (v/v) to determine the maximum level tolerated without impairing microbial growth. The CL used in this study corresponded to the liquid fraction derived from composting piles of grape-processing residues and by-products. Samples were collected from four composting piles with maturation times ranging from 1 to 4 years. The composting process was conducted by Beifiur LTDA (Garibaldi, Brazil), which also supplied the CL.
(c)
Optimization of ACM component concentrations (Step III): In the final stage, the concentrations of ACM components (based on Step I) were optimized considering the CL concentration determined in Step II. Fermentation was performed using ACM formulations containing the standard concentration (12 g/L; from Step I), half (6 g/L), and double (24 g/L). This step aimed to determine whether bacterial growth could be enhanced by adjusting the nutrient concentration.
Spent yeast and composting leachate were characterized for total organic carbon (TOC) and total Kjeldahl nitrogen (TKN) [34] to evaluate the C/N ratio of each formulation. In addition, both substrates were analyzed for essential element concentrations using atomic absorption spectroscopy (PG Instruments, Wibtoft, UK, AA500), as well as for pH and moisture content. All analyses were performed following the standards established by the Brazilian Ministry of Agriculture, Livestock, and Supply for agricultural input registration [34].
Based on the results of Step III, the bioinput was produced. The ACM components were placed in 250 mL Erlenmeyer flasks (working volume: 150 mL) and sterilized at 121 °C for 30 min. After sterilization, the flasks were inoculated with 3% (v/v) of the strain inoculum previously grown in Luria–Bertani medium. Fermentation was carried out in a shaker incubator (SL-222, Solab) at 28 °C and 160 rpm for 48 h. Colony-forming unit (CFU) counts were determined using the serial dilution method [35]. Plates were incubated in a bacterial incubator (SL-101, Solab) at 28 °C for 72 h, after which the bacterial concentration (CFU/mL) of the bioinput was quantified. This process yielded a bioinput based on an actinomycete grown in a medium formulated with winery-spent yeast and grape waste composting leachate.

2.4. Evaluation of the Plant Growth-Promoting Capacity of the Bioinput

The plant growth-promoting capacity of the developed bioinput was evaluated through pot experiments using soybean and wheat as test crops. The assays were conducted in a growth chamber at the seedling stage (15 days after germination) to assess the bioinput’s potential to enhance early plant growth parameters.
A completely randomized experimental design was employed, with six treatments, each replicated five times. Treatments included five bioinput dosages (T10: 10 mL/ha, T50: 50 mL/ha, T100: 100 mL/ha, T500: 500 mL/ha, and T1000: 1000 mL/ha) and a control (T0: no treatment). The bioinput was applied via seed treatment, with seeding rates of 60 kg/ha for soybean and 120 kg/ha for wheat. Five treated seeds were sown per pot, each containing 300 g of soil. After seven days, two seedlings were thinned, leaving three per pot for the remainder of the experiment. The pots were maintained in a growth chamber at 22–24 °C, with a 12-h light/dark cycle, and irrigated according to crop requirements.
Several seedling phytometric parameters were measured, including stem diameter (SD), shoot height (SH), shoot dry mass (SDM), root dry mass (RDM), Dickson Quality Index (DQI), total root length (TRL), root surface area (RSA), root volume (RV), length of very fine roots (LVFR), length of fine roots (LFR), and length of coarse roots (LCR). SD and SH were measured using a digital caliper and a ruler, respectively. SDM and RDM were determined after drying the samples at 65 °C in a forced-air oven. The DQI was calculated according to Equation (1) [36].
DQI = (SDM + RDM)/((SH/SD) + (SDM/RDM))
Root morphological parameters (TRL, RSA, RV, LVFR, LFR, and LCR) were evaluated using a root scanner (Epson Expression 10000XL, Seiko Epson Corporation, Nagano, Japan), and the images were analyzed with WinRHIZO software (Arabidopsis LA2400, Regent Instruments Inc., Sainte-Foy, QC, Canada). The results for phytometric parameters were expressed as mean values for each treatment.

2.5. Data Treatment

The mixture design was analyzed to determine the optimal ACM formulation using Statistica 7.0 software. The other experiments were evaluated by one-way analysis of variance (ANOVA), and differences between treatments were assessed using Tukey’s test at a 95% confidence level (p < 0.05). Prior to statistical analysis, data normality was verified to determine whether parametric or non-parametric tests should be applied. As the data followed a normal distribution, ANOVA followed by Tukey’s test was used.

3. Results and Discussion

3.1. Identification of Isolates and Evaluation of Their Agricultural Potential In Vitro

Following the pretreatment applied during the isolation stage and the selection of Gram-positive bacteria, 11 isolates were identified as actinomycetes through 16S rRNA analysis. Table 1 presents the taxonomic identification of these isolates along with the values obtained for siderophore and indolic compound production.
Among the isolates obtained from grape waste composting piles, ten strains were identified as Streptomyces spp. and one as Rhodococcus sp. All identified actinomycetes exhibited sequence similarity above 98%, which is considered the minimum threshold for confirming genus-level classification using the technique employed (Table 1).
Streptomyces sp. BEI-18A was the strain selected for further bioinput development due to its high siderophore production capacity. This strain removed 63.6% of the iron from the assay dye (Table 1). Siderophores are low-molecular-weight molecules that chelate and transport Fe3+ ions [37,38]. In agriculture, iron is an essential element for plant growth, playing catalytic roles in DNA and RNA synthesis, photosynthesis, and the production of secondary metabolites [37,39,40]. However, under neutral pH and aerobic conditions, iron becomes poorly available to plants because it is converted into insoluble oxides and hydroxides (Fe3+). In such environments, siderophores are critical, as they bind Fe3+, solubilize it, and facilitate its reduction to Fe2+, the bioavailable form absorbed by plants [41]. This process enhances nutrient uptake and stimulates plant growth. Rungin et al. [42] demonstrated this effect in rice (Oryza sativa) inoculated with a siderophore-producing Streptomyces strain compared with a genetically modified variant lacking siderophore production. The results showed greater root and shoot length, as well as increased biomass, in plants exposed to siderophores. Beyond growth promotion, siderophores also function as biocontrol agents: by sequestering iron, they deprive pathogenic microorganisms of this essential nutrient, inhibiting their proliferation and reducing disease incidence [43].
The BEI-18A strain was identified as Streptomyces sp., with Streptomyces poriferorum P01-B04 as its closest relative, showing 99.06% similarity (Table 1). S. poriferorum was first described by Sandoval-Powers et al. [44], who isolated strains from marine sponges in Norway. To date, this remains the only published report of this species. The strain is deposited in GenBank under the accession number JAELVH000000000, where it is described as having a G + C content of 70.75%, encoding an average of 7369 proteins, and a total genome length of 8.97 Mb (equivalent to 8.97 million nucleotides) [31]. The specificity of BEI-18A and its high siderophore production capacity justified its selection as the focus of this study.

3.2. Development of the Alternative Culture Medium (ACM)

The spent yeast (SY) contained 20.30% total organic carbon (TOC), 2.52% total Kjeldahl nitrogen (TKN), and had a pH of 3.62 (Table S1, Supplementary Material). Spent yeast is generally rich in diverse nutrients that support microbial growth [45,46]; however, its composition can vary depending on grape variety and processing techniques [46,47]. Paradelo et al. [48] analyzed winery-derived SY and reported 513 g/kg of TOC and 26 g/kg of total nitrogen, whereas Bustamante et al. [47] observed 300 g/kg of TOC and 35.2 g/kg of total nitrogen in a mixture of spent yeast from wineries and distilleries.
In this study, SY was one of the components tested for formulating an alternative culture medium for the cultivation of Streptomyces sp. BEI-18A using a mixture design. The results of colony-forming unit counts obtained in Step I of the design are presented in Table 2.
The p-values of the pseudo-components from the regression of the three models were analyzed (Table S2, Supplementary Material). Comparison of the models revealed that the interaction effects were not significant (p > 0.05). However, the interactions between ammonium sulfate and sucrose (X2 × X3) and between ammonium sulfate and soybean extract (X2 × X4) exhibited p-values close to significance. When the other interactions (X1 × X2, X1 × X3, X1 × X4, X3 × X4) were excluded and the analysis repeated, these interactions became significant (p < 0.05), and the quadratic model reached significance (p = 0.028; Table S3, Supplementary Material). This adjustment also increased both R2 and adjusted R2 values, supporting this decision.
Validation of the statistical model, conducted using the DOE approach, was confirmed by the F-value obtained from ANOVA exceeding the critical tabulated F-value, with the coefficient of determination (R2) further supporting the model’s adequacy. Accordingly, the model was predictive, enabling the assessment of the effects of the studied variables on the final bacterial concentration. It is important to note that, in mixture designs, the primary focus lies on the significance of the effects rather than solely on the overall R2, which reinforces the reliability of the results obtained. Based on the regression coefficients, the quadratic model derived from the mixture design is presented in Equation (2).
Bacterial concentration (log CFU/mL) = (0.583 × X1) + (3.660 × X2) + (0.598 × X3) + (0.634 × X4) − (0.548 × X2 × X3) − (0.573 × X2 × X4)
where X1: spent yeast (SY); X2: ammonium sulfate (AS); X3: sucrose (SC); X4: soybean extract (SE).
Taking into account the effects of the variables, along with considerations of waste valorization and cost reduction compared with other commercial components of the culture medium, the formulation of the ACM in Step I was established as 6 g/L of spent yeast, 4 g/L of sucrose, and 2 g/L of soybean extract. This corresponds to the minimum levels for SC, SE, and AS and the maximum level for SY, representing 50% of the formulated ACM. The C/N ratio of this formulation was 12.36, which is higher than values previously reported in the literature for Streptomyces spp. [49,50,51].
The higher C/N ratio is primarily attributed to the composition of spent yeast, which contains approximately 20% carbon and only 2.53% nitrogen (Table S1, Supplementary Material). According to the formulation, supplementation with an inorganic nitrogen source (ammonium sulfate) is not required, as its addition increases the cost of the medium and its removal does not alter the C/N ratio. In contrast, soybean extract, an organic nitrogen source containing 42.80% C and 7.99% N, is a more cost-effective nitrogen source than ammonium sulfate. In addition, soybean extract provides essential micronutrients, including 53.3 mg/kg iron (Fe), 17.0 mg/kg manganese (Mn), 0.19% total calcium (Ca), and 0.54% total phosphorus (P). The influence of the maximum level of spent yeast is also evident, as its increase is directly proportional to the rise in viable cell counts of the target microorganism.
After determining the optimal formulation for the ACM through mixture design (Step I), composting leachate was incorporated into the ACM to evaluate its effects on microbial growth (Step II). Figure 2 shows the concentration of Streptomyces sp. BEI-18A in ACM supplemented with different concentrations of grape waste composting leachate.
The addition of 2.5%, 5%, and 10% (v/v) composting leachate to the ACM significantly increased microbial concentration (p < 0.05). The presence of micronutrients such as phosphorus (0.104%), potassium (0.568%), and sulfur (1.01%), among others, supports nucleic acid synthesis, contributes to energy production, and helps maintain cellular pH, thereby enhancing cell replication [52]. For instance, iron acts as a cofactor for several enzymes and is involved in the metabolism of regulatory proteins associated with siderophore biosynthesis [53].
The composting leachate exhibited a pH of 8.98, with TOC and TKN concentrations of 0.90% and 0.13%, respectively (Table S1, Supplementary Material). Its composition varies depending on the source material [54]. Importantly, composting leachate can serve as a substrate for microbial cultivation [25], making it a promising component of an alternative culture medium for microbial growth, as it both adds value to the effluent and reduces treatment costs. When incorporated into a microbial medium, it also decreases production costs and minimizes potable water use.
In contrast, composting leachate concentrations above 25% significantly reduced the concentration of Streptomyces sp. BEI-18A (p < 0.05) compared with the control (0% leachate). Higher concentrations may promote the solubilization of additional compounds in the ACM. Since the carbon and nitrogen contributions of composting leachate are both below 1%, this solubilization may limit the availability of essential nutrients, thereby hindering microbial growth [25]. Supplementation with spent yeast is therefore necessary to meet the nutritional requirements of Streptomyces sp.
In addition, metals present in composting leachate may interact with microbial cells through ionic interactions with functional groups such as amines, hydroxyls, and carboxyls on the cell surface. These interactions can facilitate metal cation transport into the cell, potentially exerting toxic effects [55]. Elevated chlorine concentrations can also damage the cell membrane by oxidizing non-covalent and ionic interactions, hydrogen bonds, and hydrophobic bonds that maintain structural integrity. The breakdown of these bonds causes leakage and inhibits bacterial growth [56]. As composting leachate concentration increases in the culture medium, chlorine levels also rise, intensifying ionic disruption and reducing microbial concentration. Furthermore, composting leachate may contain complex organic compounds, such as humic acids, fulvic acids, and polyphenols, that inhibit microbial growth. Dilution lowers the concentration of these inhibitory substances [25,54].
Based on the microbial growth results from Step II, 10% composting leachate was incorporated into the ACM. This approach adds value to grape residue composting leachate while reducing potable water consumption in Streptomyces sp. BEI-18A cultivation. Finally, Step III was performed to establish the final ACM formulation (Figure 3).
A decrease in ACM nutrient concentration from 12 g/L to 6 g/L (Figure 3) did not result in a significant difference in microbial growth (p > 0.05). From a cost-optimization perspective, it is advantageous that lower nutrient levels can sustain microbial growth comparable to ACMs formulated with higher amounts of SY, SC, and SE. Sari et al. [57] cultivated Streptomyces spp. in an ACM and applied it to experimental soybean cultivation, where they observed enhanced plant growth and inhibition of Fusarium oxysporum. Thus, Streptomyces spp. are promising microorganisms for secreting secondary metabolites that promote plant growth and/or suppress phytopathogens, reinforcing their potential for agricultural applications.
In contrast, the use of ACM at 24 g/L negatively affected microbial growth, likely due to the final composition of the medium (Figure 3). As component concentrations increase, the solute content in the solution also rises, including sodium present at 100 mg/kg in SY, which may lead to excessive osmotic pressure. The resulting osmotic stress can destabilize cell membranes and cause cellular dehydration [53]. Another contributing factor may be the high nutrient availability at the onset of fermentation, which accelerates dissolved oxygen consumption as sugars in the ACM are oxidized to organic acids. Consequently, oxygen becomes a limiting factor for microbial growth, leading to reduced cell concentration [58,59].
Based on these findings, the optimal ACM formulation for the growth of Streptomyces sp. BEI-18A consisted of 3 g/L spent yeast, 2 g/L sucrose, and 1 g/L soybean extract, supplemented with 10% (v/v) composting leachate. The resulting culture medium exhibited a carbon-to-nitrogen (C/N) ratio of 12.36 and contained 18.31 mg/L phosphorus (P), 2.20 mg/L calcium (Ca), 4.59 mg/L iron (Fe), and 0.03 mg/L manganese (Mn), based on the composition of its components. Streptomyces sp. BEI-18A was then cultivated using this optimized formulation, yielding an actinomycete-based bioinput, which was subsequently evaluated for its potential as a plant growth promoter.

3.3. Plant Growth Promotion by Thef Streptomyces sp. BEI-18A-Based Bioinput

Several studies have demonstrated that Streptomyces spp. promote plant growth and soil health through multiple mechanisms. These include the production of phytohormones that stimulate plant development; the solubilization of minerals such as phosphorus, thereby enhancing nutrient availability; the secretion of siderophores that improve the uptake of essential nutrients; the production of antimicrobial compounds with strong antifungal activity, which protect plants against biotic stresses and contribute to disease resistance [60,61,62,63,64]. Collectively, these mechanisms enhance plant vigor and accelerate growth.
Given these effects, the analysis of root morphology is particularly relevant due to the critical role of roots in water uptake and nutrient acquisition, which are essential for supporting plant growth. Root morphological traits also regulate plant development, contributing to growth performance and adaptation to environmental conditions [65,66]. Streptomyces spp. can enhance root development by increasing mycorrhizal colonization or improving nodulation frequency, depending on the crop under evaluation [67]. Table 3 presents the results of phytometric parameters of soybean and wheat treated with Streptomyces sp. BEI-18A-based bioinput.
The root system is influenced by the soil’s supply and distribution of nutrients, which determine root complexity and total root length (TLR) [68]. In soybean crops, the highest TLR was observed in the T10 treatment (Table 3), showing a significant improvement over the control (p < 0.05). In contrast, higher doses (500 and 1000 mL/ha) reduced root length, suggesting that excessive applications may inhibit soybean root development. For wheat, the highest tested dose (1000 mL/ha) had a deleterious effect on TLR.
Stimulation of root elongation is often accompanied by increased branching, which expands the root surface area and facilitates more efficient uptake of water and nutrients [69]. In this context, root surface area (RSA) and root volume (RV) are critical traits, as both directly influence soil exploration and resource acquisition [70]. The T10 treatment increased RSA and RV in soybean compared with the control, while T100 also significantly enhanced RV (p < 0.05). These findings suggest that low to moderate doses of the Streptomyces-based bioinput can improve root surface development and volume, thereby enhancing nutrient absorption. Root diameter is another important trait, as thinner roots penetrate soil more effectively and capture nutrients more efficiently than thicker roots [71]. Consequently, plants with a higher proportion of very fine roots typically exhibit improved nutrition, survival, and maturity.
Application of the Streptomyces-based bioinput did not significantly affect the length of very fine roots in soybeans. However, T10 improved fine root development compared with T0, T500, and T1000 (p < 0.05), while T10 and T50 enhanced coarse root development relative to the control (p < 0.05). Increasing coarse root formation is critical, as these roots provide structural support. In wheat, no significant differences in root diameter traits were observed among treatments.
In soybeans, T10 exhibited the best overall performance across root parameters, with the exception of very fine root length (LVFR). In wheat, no clear optimal treatment was observed, likely due to crop-specific responses. These results highlight the potential of Streptomyces-based bioinputs to enhance root development traits that are critical for plant nutrition and productivity. They also emphasize the importance of dosage, as excessive applications negatively affected early plant growth in both crops. These findings align with Tian et al. [64], who reported a non-linear response to Streptomyces-based biofertilizers: in wheat, the optimal dose (S10) increased root activity by 44.21% and dry weight by 26.98%, while higher doses reduced benefits. Similarly, Nivedita et al. [72] demonstrated strong growth-promoting potential under abiotic stress but noted multifactorial limitations. Reduced efficacy at high doses may result from toxic metabolite accumulation or competition with native soil microbes.
Other phytometric parameters were used to calculate the Dickson Quality Index (DQI), a robust metric for evaluating plant quality during early vegetative growth [73]. DQI integrates several morphological traits, including shoot length, stem diameter, and the dry mass of shoots and roots, and is an indicator of seedling survival and establishment potential. In soybeans, T10, T50, T100, and T500 yielded the highest DQI values, with T50 and T100 performing significantly better than the control (p < 0.05, Table 3). In wheat, T100 produced the highest DQI values, significantly outperforming T0 and T1000 (p < 0.05). Conversely, both the control and T1000 recorded the lowest DQI values, underscoring the dual potential of the bioinput to either promote or impair early crop development depending on dosage.
The superior performance of certain treatments over the control can be attributed to the ability of Streptomyces spp. to enhance nutrient availability, produce plant growth-promoting compounds, and mitigate biotic and abiotic stresses [7,67]. Overall, the Streptomyces sp. BEI-18A-based bioinput positively influenced DQI and root morphology in soybean and wheat seedlings. Gopalakrishnan et al. [74] similarly reported improvements in root morphology and yield in sorghum and rice, linked to phytohormone production (e.g., indole acetic acid), siderophores, and chitinase. Wang et al. [75] confirmed the dual role of Streptomyces aureovercitillatus HN6 as a plant growth promoter and biocontrol agent, increasing germination rates, root development, aboveground biomass, and disease resistance under both controlled and field conditions. These effects were attributed to phytohormone production, nutrient solubilization, siderophore synthesis, and antimicrobial activity. Similar conclusions were reported by Domínguez-González et al. [76], Gao et al. [77] and Htwe et al. [78] who emphasized the economic and environmental sustainability of Streptomyces-based biofertilizers. Kaur et al. [63] compared three Streptomyces strains (WR27, WR15, and WS6) with a standard bioinoculant, applied individually or in combination with Azotobacter. Strains WR27 and WR15 showed superior agronomic performance, particularly when combined with Azotobacter. The WR27 + Azotobacter treatment (T6) achieved the highest grain yield (5.7 t/ha), surpassing both the control (5.0 t/ha) and a commercial consortium (Azotobacter, Bacillus, and Pseudomonas). It also improved plant height, biomass, spike traits, and grain weight. These findings demonstrate that combining only two microorganisms can outperform existing formulations, underscoring the potential of WR27 for wheat biofertilization.
The cultivation of Streptomyces sp. BEI-18A in an alternative culture medium formulated with winery spent yeast and composting leachate represents a sustainable strategy for bioinput development. This approach is particularly relevant in Brazil, where no Streptomyces-based bioinput has yet been registered, highlighting a major technological gap. Beyond enhancing crop performance, this formulation advances several Sustainable Development Goals (SDGs). It contributes to SDG 2 (Zero Hunger) by increasing agricultural productivity, while the valorization of agro-industrial residues supports SDG 12 (Responsible Consumption and Production) and SDG 9 (Industry, Innovation, and Infrastructure). The reduction in fertilizer and pesticide use aligns with SDG 15 (Life on Land) and SDG 14 (Life Below Water), while replacing potable water with composting leachate directly addresses SDG 6 (Clean Water and Sanitation). Moreover, the overall reduction in environmental impacts strengthens SDG 13 (Climate Action). Collectively, these outcomes highlight the potential of actinomycete-based bioinputs to promote sustainable agriculture within a circular bioeconomy framework. The innovation and sustainability aspects of the bioinput, related to the development of an alternative culture medium for the bioprospected actinomycete, are illustrated in Figure 4, highlighting its alignment with several Sustainable Development Goals (SDGs).

4. Conclusions

In this study, actinomycetes were isolated from grape waste composting piles and evaluated for their agricultural potential through the production of siderophores and indolic compounds. Streptomyces sp. BEI-18A was identified as the most promising strain due to its high siderophore production. An alternative culture medium (ACM) formulated with winery-spent yeast and composting leachate was developed to cultivate this actinomycete and produce a sustainable agricultural bioinput. Based on microbial growth performance, the final ACM composition was defined as 3 g/L spent yeast, 2 g/L sucrose, 1 g/L soybean extract, and 10% (v/v) composting leachate. This formulation yielded a Streptomyces sp. BEI-18A-based bioinput with a concentration of 7.8 × 107 CFU/mL (7.98 log CFU/mL). The bioinput enhanced several phytometric parameters of soybean and wheat, particularly at lower dosages (10, 50, and 100 mL/ha), with plant responses varying depending on the crop tested.
The inclusion of winery-spent yeast and grape waste composting leachate as ACM components contributes to the valorization of residues and effluents while reducing culture medium costs and minimizing potable water use in the fermentation process. This strategy integrates circular economy principles into bioinput production, offering a promising pathway toward more sustainable agriculture. Consequently, the Streptomyces sp. BEI-18A-based bioinput shows strong potential for commercialization in the Brazilian agricultural sector, supported by its plant growth-promoting effects and the current absence of registered actinomycete-based bioinputs for agricultural use in Brazil.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation11100556/s1, Table S1: Characterization of winery spent yeast and grape waste composting leachate; Table S2: p-value of pseudo-components resulting from the mixture design; Table S3: Analysis of variance (ANOVA) of the models resulting from the analysis of the mixture design (Step I).

Author Contributions

Conceptualization: M.T.N.; Formal analysis and investigation: M.T.N., A.R., V.A.S., B.S.M., D.D.C.K. and C.V.; Writing—original draft preparation: M.T.N., A.R., V.A.S., B.S.M., D.D.C.K., C.V., H.T. and V.F.; Writing—review and editing: M.T.N., H.T., V.F., L.M.C. and J.S.P.; Supervision: L.M.C. and J.S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed in part by the National Council for Scientific and Technological (CNPq, Brazil) (Project Codes 403658/2020-9 and 140541/2021-7) and Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) (Finance Code 001).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Methodological steps performed in this study.
Figure 1. Methodological steps performed in this study.
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Figure 2. Streptomyces sp. BEI-18A growth after 48 h in the ACM with different concentrations of composting leachate. Different letters indicate a statistical difference (p < 0.05) according to Tukey’s test.
Figure 2. Streptomyces sp. BEI-18A growth after 48 h in the ACM with different concentrations of composting leachate. Different letters indicate a statistical difference (p < 0.05) according to Tukey’s test.
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Figure 3. Growth of Streptomyces sp. BEI-18A after 48 h at different final concentrations of ACM components (Step I) supplemented with 10% (v/v) composting leachate (Step II). Different letters indicate a statistical difference (p < 0.05) according to Tukey’s test.
Figure 3. Growth of Streptomyces sp. BEI-18A after 48 h at different final concentrations of ACM components (Step I) supplemented with 10% (v/v) composting leachate (Step II). Different letters indicate a statistical difference (p < 0.05) according to Tukey’s test.
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Figure 4. Contribution of the Streptomyces sp. BEI-18A-based bioinput to the UN Sustainable Development Goals (SDGs).
Figure 4. Contribution of the Streptomyces sp. BEI-18A-based bioinput to the UN Sustainable Development Goals (SDGs).
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Table 1. Taxonomic identification and production of siderophores and indolic compounds by actinomycete isolates.
Table 1. Taxonomic identification and production of siderophores and indolic compounds by actinomycete isolates.
StrainIdentificationClosest SpeciesFragment Size (pb)Similarity (%)Siderophores (%)Indolic Compounds (µg mL−1)
BEI-02AStreptomyces sp.S. thermocoprophilus NBRC 10077178598.8538.515.9
BEI-06AStreptomyces sp.S. poriferorum P01-B0475599.7464.00
BEI-07AStreptomyces sp.S. albus NRRL B-181176510026.54.2
BEI-11AStreptomyces sp.S. speibonae PK-Blue79098.9900
BEI-12AStreptomyces sp.S. spiralis NBRC 142172899.8620.61.5
BEI-13ARhodococcus sp.R. qingshengii JCM 1547774110000
BEI-16AStreptomyces sp.S. poriferorum P01-B0474499.6038.92.6
BEI-17AStreptomyces sp.S. malaysiense MUSC 13673399.7041.15.6
BEI-18AStreptomyces sp.S. poriferorum P01-B0464199.0663.65.0
BEI-19AStreptomyces sp.Streptomyces sp.55098.3616.50
BEI-22AStreptomyces sp.S. thermocoprophilus B1977899.4911.810.4
Table 2. Streptomyces sp. BEI-18A concentration (Log CFU/mL) in different ACM formulations.
Table 2. Streptomyces sp. BEI-18A concentration (Log CFU/mL) in different ACM formulations.
ExperimentSY (g/L)AS (g/L)SC (g/L)SE (g/L)C/NBacterial Concentration
(Log CFU/mL) *
16.000.004.002.0012.367.13 ± 0.25
24.002.004.002.004.666.57 ± 0.67
34.000.006.002.0016.977.22 ± 0.06
44.000.004.004.0010.087.40 ± 0.19
55.001.004.002.006.977.10 ± 0.43
65.000.005.002.0014.446.99 ± 0.21
75.000.004.003.0011.087.22 ± 0.27
84.001.005.002.007.866.33 ± 0.64
94.001.004.003.006.576.48 ± 0.67
104.000.005.003.0012.777.09 ± 0.17
114.650.654.652.009.007.11 ± 0.01
124.650.654.002.657.906.39 ± 0.12
134.650.004.652.6512.657.25 ± 0.42
144.000.654.652.658.566.72 ± 0.17
156.000.004.002.0012.366.69 ± 0.12
Legend: * Mean ± SD (n = 2); SY: spent yeast; AS: ammonium sulfate; SC: sucrose; SE: soybean extract, C/N: carbon:nitrogen ratio, CFU/mL: colony-forming units per mL, Log: logarithmic function of CFU/mL.
Table 3. Phytometric parameters of soybean and wheat under different treatments with Streptomyces sp. BEI-18A-based bioinput.
Table 3. Phytometric parameters of soybean and wheat under different treatments with Streptomyces sp. BEI-18A-based bioinput.
TreatmentTLR
(cm)		RSA
(cm2)		RV
(cm3)		LVFR (cm)LFR
(cm)		LCR
(cm)		SH
(cm)		SD
(mm)		SDM
(g)		RDM
(g)		DQI
SoybeanT064.17 a23.33 a0.61 a15.36 a42.48 a6.33 a9.26 a1.93 a0.347 ab0.090 a0.051 ab
T1099.34 b38.70 b1.22 c20.21 a66.14 b12.97 c11.08 a1.99 a0.353 ab0.148 b0.063 bc
T5086.75 ab34.40 ab1.09 bc20.81 a55.66 ab12.28 c12.04 a2.01 a0.372 b0.164 b0.065 c
T10070.45 ab27.27 ab0.86 abc15.55 a45.94 ab8.96 ab10.59 a2.09 a0.361 ab0.147 b0.068 c
T50062.80 a22.50 a0.66 a15.94 a39.02 a7.84 ab9.40 a2.09 a0.348 ab0.113 a0.062 bc
T100064.79 a24.16 a0.72 ab14.63 a42.52 a7.63 a11.66 a1.92 a0.342 a0.102 a0.047 a
WheatT022.17 ab5.36 ab0.107 a7.59 a11.25 a0.81 a15.99 a0.72 a0.035 a0.039 bc0.0030 a
T1025.60 ab6.64 b0.137 a9.99 a11.89 a1.15 a19.32 a0.74 a0.085 b0.049 c0.0048 bc
T5019.90 ab4.91 ab0.103 a6.05 a10.69 a1.02 a15.59 a0.76 a0.057 ab0.033 abc0.0040 bc
T10025.10 ab6.18 ab0.130 a12.91 a12.62 a0.75 a15.72 a0.82 a0.085 b0.035 bc0.0056 c
T50027.95 b5.78 ab0.097 a15.90 a11.09 a0.93 a15.29 a0.81 a0.071 ab0.030 ab0.0047 bc
T100014.26 a4.05 a0.103 a6.85 a8.26 a1.06 a16.05 a0.71 a0.049 ab0.017 a0.0032 ab
Legend: T0: control (no bioinput application); T10: 10 mL/ha; T50: 50 mL/ha; T100: 100 mL/ha; T500: 500 mL/ha; T1000: 1000 mL/ha; TLR: total length of the roots; RSA: root surface area; RV: root volume; LVFR: length of very fine roots; LFR: length of fine roots; LCR: length of coarse roots; SH: shoot height; SD: stem diameter; SDM: shoot dry mass; RDM: root dry mass; DQI: Dickson Quality Index. Different letters in the column indicate a significant difference (p < 0.05) between the treatments performed by crop.
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Nazari, M.T.; Rubert, A.; Schommer, V.A.; Machado, B.S.; Vancini, C.; Krein, D.D.C.; Ferrari, V.; Treichel, H.; Colla, L.M.; Piccin, J.S. From Isolation to Plant Growth Evaluation: Development of a Streptomyces-Based Bioinput Using Spent Yeast and Composting Leachate. Fermentation 2025, 11, 556. https://doi.org/10.3390/fermentation11100556

AMA Style

Nazari MT, Rubert A, Schommer VA, Machado BS, Vancini C, Krein DDC, Ferrari V, Treichel H, Colla LM, Piccin JS. From Isolation to Plant Growth Evaluation: Development of a Streptomyces-Based Bioinput Using Spent Yeast and Composting Leachate. Fermentation. 2025; 11(10):556. https://doi.org/10.3390/fermentation11100556

Chicago/Turabian Style

Nazari, Mateus Torres, Aline Rubert, Vera Analise Schommer, Bruna Strieder Machado, Camila Vancini, Daniela Dal Castel Krein, Valdecir Ferrari, Helen Treichel, Luciane Maria Colla, and Jeferson Steffanello Piccin. 2025. "From Isolation to Plant Growth Evaluation: Development of a Streptomyces-Based Bioinput Using Spent Yeast and Composting Leachate" Fermentation 11, no. 10: 556. https://doi.org/10.3390/fermentation11100556

APA Style

Nazari, M. T., Rubert, A., Schommer, V. A., Machado, B. S., Vancini, C., Krein, D. D. C., Ferrari, V., Treichel, H., Colla, L. M., & Piccin, J. S. (2025). From Isolation to Plant Growth Evaluation: Development of a Streptomyces-Based Bioinput Using Spent Yeast and Composting Leachate. Fermentation, 11(10), 556. https://doi.org/10.3390/fermentation11100556

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