Circular Approach in Development of Microbial Biostimulants Using Winery Wastewater

Abstract

Sustainable development requires implementation of eco-friendly practices and a circular approach in both agricultural and industrial systems. This study evaluated winery flotation wastewater (WFW) as a cultivation substrate for Bacillus sp. 10/R isolated from grapevine rhizosphere for sustainable biostimulant production. The bacterial isolate was characterized by 16S rRNA sequencing and biochemical tests, showing the highest similarity with Bacillus mojavensis and Bacillus halotolerans. Plant growth-promoting traits were assessed via assays for hydrolytic enzymes, ACC (1-aminocyclopropane-1-carboxylate) deaminase, and IAA (indole acetic acid) production, as well as for phosphate solubilization. The isolate was cultivated in WFW, including monitoring of biomass growth, enzymatic activity, and substrate composition changes. The resulting cultivation broths based on WFW (WFW-CB) and nutrient broth (NB-CB) were tested as barley seed treatment at five dosages, using sterile media and water as controls. The results have displayed strong pectinase (EAI–enzyme activity index 2.79) and cellulase activity (2.33), moderate xylanase (1.75) and ACC deaminase activity (growth zone 54.67 ± 0.58 mm), and moderate IAA production (9.66 µg/mL). Biomass content has increased by two log units within 48 h (up to 9.06 log CFU/mL), with stable pectinase activity (~2.2 U/mL). Germination assays revealed that 10% WFW-CB and 50% WFW enhanced germination indices and biomass, whereas undiluted WFW and WFW-CB inhibited germination. These results indicate that WFW is a suitable substrate for Bacillus sp. 10/R cultivation, linking industrial wastewater valorization with plant biostimulant production in a circular economy framework.

1. Introduction

Although the use of chemical fertilizers and pesticides has significantly enhanced agricultural productivity over the past five decades, their excessive application has also contributed to environmental issues, including groundwater contamination, soil degradation, and biodiversity loss [1,2,3]. Therefore, it is essential to develop innovative, environmentally sustainable agricultural practices that can maintain high productivity while mitigating the adverse impacts of climate change and environmental degradation due to chemical pollution [4]. Green biotechnological products represent one of the promising solutions in this field. In this context, plant growth-promoting (PGP) bacteria have emerged as key players in the development of microbial-based products intended for sustainable agricultural use [5,6]. These beneficial microorganisms exhibit a variety of direct and indirect mechanisms, including nitrogen fixation, nutrient solubilization, phytohormone production, synthesis of hydrolytic enzymes, antibiotics and siderophores, as well as induction of systemic resistance in plants [7,8]. Owing to these diverse activities, PGP bacteria-based products can be effectively utilized as biofertilizers, biostimulants, biocontrol agents, and soil quality enhancers, providing a promising tool to improve crop productivity within sustainable agricultural systems [9,10].

Among PGP microorganisms, including wide range of bacterial and fungal species such as Azotobacter, Azospirillum, Rhizobium, Pseudomonas, Trichoderma, etc., Bacillus species stand out as the most studied and utilized in sustainable agriculture due to their diverse functional traits and adaptability to various environmental conditions. These rhizobacteria are recognized for their ability to enhance plant growth and resilience to abiotic stresses, while also providing biological control potential against plant pathogens [11,12]. A key advantage of Bacillus spp. is their ability to form endospores, which ensures high environmental stability and viability during formulation, storage and field application, making them particularly suitable for large-scale use in agricultural settings [13,14]. Application of various formulations of Bacillus-based plant biostimulants has been investigated across the wide range of crops and testing conditions (including seed germination experiments, growth chambers, greenhouse and field experiments), indicating their contributions to improved plant growth and crop quality, resilience and advanced immune responses of plants under abiotic and biotic stresses [15,16,17].

However, despite the promising potential of microbial-based bioagents, including Bacillus spp., their wider application as biofertilizers in agricultural production systems is still limited, mainly due to the high costs associated with biotechnological production processes. In this regard, the utilization of alternative, complex media derived from industrial effluents represents a promising and underexplored approach for the cost-effective production of microbial biocontrol agents/plant biostimulants [18]. Such media would not only contribute to the economic feasibility of bioprocesses but also align with the principles of circular economy and industrial symbiosis by valorizing waste streams while reducing freshwater consumption [19,20].

The wine industry is considered one of the most significant socio-economic activities in Europe [21]. According to the International Organization of Vine (OIV), the global vineyard surface area in 2024 was reported at 7.1 million hectares, while global wine production reached approximately 226 million hectoliters [22]. Processing 1000 kg of grapes typically results in 750 L of wine, alongside 1650 L of wastewater and about 200 kg of solid residues, which present a significant environmental burden due to their high organic load and the presence of volatile organic compounds [23]. However, the rich organic composition of these waste streams also indicates their potential for valorization through biotechnological approaches, particularly microbial bioconversion, offering opportunities for the development of sustainable processes within the wine industry [24]. The utilization of winery waste for the cultivation of Bacillus spp. provides an opportunity to reduce environmental pollution while simultaneously producing biofertilizers and plant growth-promoting agents, aligning with the circular economy principles [14].

The aim of this study was to evaluate the potential of Bacillus sp. 10/R, isolated from the grapevine rhizosphere, as a plant growth-promoting (PGP) agent. Specifically, the study focused on investigating the production of hydrolytic enzymes, indole-3-acetic acid (IAA), ACC deaminase, and phosphate solubilization as the prominent PGP traits. In addition, the nutritional potential of winery flotation wastewater (WFW) was assessed by its application as a growth substrate for cultivation of the aforementioned bacterial isolate. Throughout the cultivation period, biomass accumulation and enzyme activities were monitored. Finally, the cultivation broth, applied at different concentrations as seed treatment, was used to evaluate its effect on barley seed germination. The main hypothesis of the study thus was that the WFW as an industrial effluent could be applied as a bacterial growth substrate in production of Bacillus-based plant biostimulants. The proposed industrial symbiosis framework would contribute to circular utilization of winery waste in a subsequent industrial sector producing bio-based alternatives for agrochemicals aimed primarily for applications in organic agriculture, therefore improving sustainability indices of both industrial and agricultural sector.

2. Materials and Methods

2.1. Microorganisms

In this study, Bacillus sp. 10/R, isolated from the rhizosphere of Merlot grapevine (Vitis vinifera cv. Merlot), was used as the biostimulant active component. For the isolation procedure, 1 g of the collected rhizosphere sample was resuspended in 9 mL of sterile saline and incubated at 28 °C for 15 min with continuous agitation on a laboratory rotary shaker (KS 4000i control, IKA^® Werke, Staufen im Breisgau, Germany) at 150 rpm. After homogenization, selective isolation of spore-forming bacteria was achieved by subjecting the suspension to heat treatment at 100 °C for 7 min. Serial tenfold dilutions (10⁻¹ to 10⁻³) were prepared, and 500 µL of each dilution was spread onto HiChrome Bacillus agar (HiMedia Laboratories, Mumbai, India) and incubated at 28 °C for 48 h. Colonies with morphological characteristics typical for Bacillus spp. were subcultured on fresh selective media until pure cultures were obtained [25]. The pure isolate was maintained on nutrient agar slants at 4 °C, as well as a glycerol stock culture at −80 °C in the culture collection of the Laboratory for Biochemical Engineering, Faculty of Technology Novi Sad, University of Novi Sad, Serbia.

2.2. Genomic DNA Extraction and 16S rRNA Sequencing

Bacillus sp. 10/R was cultured in 20 mL of nutrient broth (HiMedia Laboratories, Mumbai, India) overnight at 28 °C with continuous agitation at 150 rpm on a laboratory rotary shaker (KS 4000i control, IKA^® Werke, Staufen im Breisgau, Germany). Cells were harvested by centrifugation at 5000 rpm for 15 min, and the resulting pellet was resuspended in 10 mL of lysis buffer containing 0.3 M sucrose, 25 mM EDTA, 25 mM Tris-HCl, and RNase (2 U), adjusted to pH 7.5. The suspension was incubated with lysozyme (10 mg) at 37 °C for 30 min, followed by the addition of proteinase K (5 mg, Sigma Aldrich–Merck, Darmstadt, Germany) and 1 mL of 10% (w/v) SDS, and incubated at 55 °C for 90 min. Subsequently, 15 mL of chloroform and 3.6 mL of 5 M NaCl were added, and the mixture was subjected to end-over-end rotation for 20 min. Cell debris were removed by centrifugation at 5000 rpm for 20 min, and DNA was precipitated from the supernatant using isopropanol (1:1, v/v). The DNA pellet was washed with 1 mL of 70% (v/v) ethanol, air-dried, and dissolved in DNA- and RNA-free water (Thermo Fischer Scientific, Waltham, MA, USA) [26].

The 16S rRNA gene was amplified from the extracted genomic DNA using universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) in a Surecycler 8800 Thermocycler (Agilent Technologies, Santa Clara, CA, USA). Amplified DNA fragments were bidirectionally sequenced (Macrogen Europe, Amsterdam, The Netherlands) with the same primers as in the amplification step. The obtained sequences were identified using the blastn feature of BLAST (Basic Local Arrangement Search Tool—https://blast.ncbi.nlm.nih.gov/, accessed on 16 July 2025) and evolutionary analyses were performed using MEGA12 software. The phylogenetic tree was generated using the maximum likelihood method and the 16S rRNA sequence of Escherichia coli NBRC 102203 was applied as an outgroup.

2.3. Biochemical Characterization of Bacillus sp. 10/R

Biochemical characterization of Bacillus sp. 10/R was performed using the commercial API test kits: API 50 CH, API 20 E, and API ZYM (bioMérieux, Marcy-l'Étoile, France), whose application was aimed at deciphering the ability of the bacterial isolate to consume various nutrient sources, produce several metabolites relevant for biochemical distinction among bacterial species, as well as to better understand enzymatic profile of the investigated strain, respectively. All tests were carried out following the manufacturer’s instructions. Briefly, bacterial cells were suspended in sterile saline solution to the recommended turbidity and inoculated into the respective API strips or galleries, containing appropriate substrates for the aforementioned biochemical reactions. The strips were incubated at 28 °C for 24 to 48 h, depending on the test requirements. After incubation, the results were recorded based on color changes or enzymatic activity indicators according to the API reading guidelines.

2.4. Screening of Plant-Growth Promotion Traits

2.4.1. Screening of Enzyme Production on Agar Plates

The enzymatic activity of Bacillus sp. 10/R isolate was screened using semi-solid media containing specific substrates for the target enzymes (pectinase, cellulase, xylanase, protease), important for complex nutrient release in agricultural applications. The incubation conditions were similar in each enzyme activity screening experiment: 120 h at 28 °C. All media were sterilized in an autoclave at 121 °C and 2.1 bar for 20 min, poured into Petri dishes, and allowed to solidify. Subsequently, plates were point-inoculated at the center using a sterile inoculating pin from an actively growing culture of the strain Bacillus sp. 10/R. After the incubation period, the diameters of the growth zones and the halo zones around the colonies were measured. For certain assays aimed at specific enzymes, appropriate reagent or indicator solutions were applied onto the surface of the media to enhance the visualization of the halo zones where needed, as explained in the following sections.

The Enzymatic Activity Index (EAI) for each tested enzyme was calculated according to the following formula [9]:

$$\mathrm{E}\mathrm{A}\mathrm{I}={\displaystyle \frac{\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}+\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o} \mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}}{\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}}}$$

(1)

Pectinase Activity

The pectinolytic activity of Bacillus sp. 10/R was assessed on a medium containing (g/L): NaNO₃ 1, KCl 1, K₂HPO₄ 1, MgSO₄·7H₂O 1, yeast extract 0.5, pectin 10, and agar 15 [27]. The pH of the medium was adjusted to 7.00 ± 0.02 prior to the addition of agar. Halo zones indicating pectinolytic activity were visualized by flooding the plates with Gram’s iodine solution for 5 min, followed by rinsing with distilled water and the measurement of the corresponding diameters of bacterial growth and halo zones.

Cellulase and Xylanase Activity

The cellulase and xylanase activity of Bacillus sp. 10/R isolate was assessed on medium containing (g/L): CMC or xylan 5, glucose 1, yeast extract 0.5, KCl 1, K₂HPO₄ 1, NaNO₃ 1, MgSO₄·7H₂O 0.5, agar 17 [28]. For improved visualization of the clear zones surrounding microbial growth, the plates were flooded with Congo red solution (0.5% w/v) for 15 min after incubation, followed by rinsing with 1 M NaCl solution for 15 min and the measurement of the corresponding diameters of bacterial growth and halo zones.

Protease Activity

The proteolytic activity of the investigated Bacillus sp. 10/R was evaluated on modified skim milk agar containing (g/L): skim milk powder (Centrohem, Stara Pazova, Serbia) 28, tryptone 5, yeast extract 2.5, glucose 1, and agar 15 [29]. The pH of the medium was adjusted to 7.00 ± 0.02 before agar was added. After the incubation under the previously described conditions, the halo zones and bacterial growth diameters were measured.

2.4.2. ACC Deaminase Activity

The ability of Bacillus sp. 10/R to produce ACC deaminase was assessed using agar-solidified DF minimal medium supplemented with 3 mM 1-aminocyclopropane-1-carboxylate (ACC—Sigma Aldrich–Merck, Darmstadt, Germany) as the sole nitrogen source, following the procedure described by Penrose and Glick (2003) [30]. Briefly, bacterial isolates were streaked onto DF minimal medium plates containing ACC and incubated at 28 °C for up to 7 days. Growth on this medium, which lacks alternative nitrogen sources, was taken as an indicator of ACC deaminase activity, since only bacteria capable of utilizing ACC as a nitrogen source are able to grow under these conditions.

2.4.3. Phosphate Solubilization Assay

Phosphate solubilization by Bacillus sp. BioSol021 was evaluated using Pikovskaya’s agar medium (Himedia Laboratories, Mumbai, India). The plates were point-inoculated at the center by transferring a small amount of biomass from an actively growing culture using a sterile inoculating pin. The plates were incubated at 28 °C for 120 h. Following incubation, the diameters of the colonies and the surrounding halo zones were measured, and the phosphate solubilization index (PSI) was calculated according to the following formula [31].

$$\mathrm{P}\mathrm{S}\mathrm{I}={\displaystyle \frac{\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}+\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o} \mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}}{\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}}}$$

(2)

2.4.4. Indole Acetic Acid Production

For indole-3-acetic acid (IAA) production assay Bacillus sp. 10/R was cultivated in nutrient broth medium (HiMedia Laboratories, Mumbai, India) supplemented with L-tryptophan (1.02 g/L) as a precursor of IAA. The cultivation was carried out in Erlenmeyer flasks at 28 °C for 48 h on a laboratory rotary shaker at 150 rpm under spontaneous aeration. The determination of IAA was performed by colorimetric method with slight modifications according to Syed-Ab-Rahman et al. (2018) [32]. Briefly, 1 mL of the culture supernatant was mixed with 2 mL of Salkowski reagent (1.2% [w/v] FeCl₃ in 7.9 M H₂SO₄) and incubated in the dark at room temperature for 30 min. The development of a pink coloration indicated the production of IAA. After incubation, the absorbance was measured at 535 nm using a UV–Vis spectrophotometer (UV 1800, Shimadzu, Kyoto, Japan). Distilled water served as a blank, and the IAA concentration in the samples was determined using a calibration curve prepared with an indole-3-acetic acid standard (Sigma-Aldrich–Merck, Darmstadt, Germany).

2.5. Cultivation Media

Inoculum of Bacillus sp. 10/R was prepared using liquid commercial medium nutrient broth (HiMedia Laboratories, Mumbai, India). The cultivation of the producing microorganisms was performed using a medium based on winery flotation wastewater (WFW). The initial quality parameters of the WFW were determined as described in the following section. Prior to use, the wastewater was diluted with distilled water in a 1:2 (v/v) ratio, and the initial pH of the cultivation medium was adjusted to 6.5 ± 0.2. The media were sterilized by autoclaving at 121 °C for 20 min under a pressure of 2.1 bar.

2.6. Physicochemical Characterization of WFW

The physicochemical characterization of WFW was conducted prior to medium inoculation and after 48 h of cultivation. To determine the basic nutritional profile of the WFW medium and the 48-h cultivation broth, several methods and techniques were used to assess dry matter, sugar content, protein and cellulose content, as well as alcohol content. The total solids of samples was determined gravimetrically, following the method described in the Official Gazette of the SFRY, No. 29/83 [33]. Soluble solids were measured refractometrically using an Abbe universal refractometer (Carl Zeiss, Oberkochen, Germany), with direct readings taken from the refractometer scale. The total sugar content in the samples was analyzed using the Luff-Schoorl method [33], while reducing sugars were quantified volumetrically according to the procedure outlined in the same source. Cellulose (crude fiber) content was determined using the Kirschner–Ganakova method, as described by Vračar (2001) [34]. The protein content was determined by the Kjeldahl method [35]. Alcohol (ethanol) content was determined by distillation, followed by oxidation with potassium dichromate in the presence of sulfuric acid. The excess dichromate was titrated with ammonium ferrous sulfate using iron-ortho-phenanthroline as an indicator, according to the Official Gazette of the SFRY, No. 29/83 [33].

2.7. Inoculum Preparation and Cultivation Media and Conditions

The inoculum of Bacillus sp. 10/R was prepared by transferring a loopful of the previously refreshed culture into sterile nutrient broth, followed by cultivation at 28 °C for 24 h on a laboratory rotary shaker at 150 rpm. The previously prepared and sterilized WFW-based medium was inoculated with 10% (v/v) of the inoculum. Cultivation was carried out at 28 °C for 96 h on a laboratory rotary shaker at 175 rpm under spontaneous aeration. Samples of the cultivation broth were collected at 24-h intervals for the determination of biomass content and enzymatic activity.

2.8. Biomass Content Determination

The droplet plate method was applied to assess biomass content of Bacillus sp. 10/R. Serial dilutions of the bacterial suspension were prepared by adding 0.1 mL of sample into 0.9 mL of sterile saline, vortexing for 8 s, and repeating to achieve a 10-fold dilution series. For plating, agar plates were divided into four quadrants, each labeled for a different dilution. After vortexing, 10 µL of each dilution was added as four evenly spaced drops (total volume of 40 µL) onto the designated area of the agar plates.

The drops were allowed to absorb (~15–20 min on pre-dried agar), after which the plates were inverted and incubated 28 °C for 17–20 h. Colonies within drops containing 3–30 colonies were counted, and the viable cell concentration was calculated as colony-forming units per milliliter (CFU/mL) using the formula [36]

$$\mathrm{C}\mathrm{F}\mathrm{U}={\displaystyle \frac{\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{e}\mathrm{s} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{d}\mathrm{r}\mathrm{o}\mathrm{p}}{0.01 \mathrm{m}\mathrm{L}}}\times \mathrm{d}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}$$

(3)

2.9. Quantitative Determination of Enzyme Activity in Liquid Culture

Pectinase activity was determined using a modified dinitrosalicylic acid (DNS) method as described by Mohandas et al. [27]. Briefly, culture broth of Bacillus sp. 10/R was centrifuged at 10,000 rpm for 10 min, and the supernatant was used as the crude enzyme extract. The reaction mixture containing 0.5 mL of culture supernatant and 0.5 mL of 1% (w/v) pectin (Sigma Aldrich–Merck, Darmstadt, Germany) as substrate was incubated at 50 °C for 30 min in a water bath. After incubation, 1.5 mL of DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. The samples were then cooled, diluted appropriately, and the absorbance was measured at 540 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The cultivation medium (sterilized WFW) without the bacteria was used as a control. Mono-D-galacturonic acid was used to prepare the standard calibration curve, and one unit of pectinase activity was defined as the amount of enzyme required to release 1 µmol of galacturonic acid per mL per minute under the assay conditions. The pectinase activity was calculated using the following formula:

$$\mathrm{P}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{\mathrm{m}\left(\mathrm{m}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{c} \mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\left(\mathrm{\mu }\mathrm{g}\right)\right)}{\mathrm{v}(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e})\times \mathrm{t}(\mathrm{i}\mathrm{n}\mathrm{c}\mathrm{u}\mathrm{b}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e})\times 212.15}}$$

(4)

Quantitative determination of endo-cellulase and endo-xylanase activities in the culture supernatants was performed using the Megazyme assay kits (K-CellG5, K-XylX6) according to the manufacturer’s instructions (Megazyme, Wicklow, Ireland). The original extracts were preprared by diluting supernatant samples in sodium phosphate buffer. For enzyme assays, 0.10 mL of substrate solution (CellG5 for cellulase, XylX6 for xylanase) was mixed with 0.1 mL of the diluted samples (original extracts) and incubated for 10 min at 40 °C. The reactions were stopped with 3.0 mL of the stopping reagent (2% (w/v) Tris buffer (pH 10)), and absorbance was measured at 400 nm (UV-1800, Shimadzu, Kyoto, Japan). Reagent blanks were prepared for both cellulase and xylanase by mixing the stopping reagent, substrate solution, and the diluted enzymes. Enzyme activity was expressed as CellG5 units/mL for cellulase and XylX6 units/mL for xylanase. One CellG5 unit is defined as the amount of enzyme required to release 1 μmol of 4-nitrophenol from CellG5 per minute, while one XylX6 unit is defined as the amount of enzyme required to release 1 μmol of 4-nitrophenol from XylX6 per minute under the assay conditions.

2.10. Seed Germination Assay

For seed germination assay barley (Hordeum vulgare, Nonius variety) seeds were used, which were commercially supplied from Institute of Field and Vegetable Crops, Novi Sad, Serbia. The seeds were surface sterilized using the chlorine bleach solution (6% (v/v), 1 min) and thoroughly washed using the sterile distilled water for 5 min [37]. After drying, ten seeds were placed in 90 mm diameter Petri dishes containing filter paper. The moisture required for germination was provided by adding 2 mL of tap water onto the filter paper. Each dish with seeds was treated with 1 mL of cultivation broth based on WFW or nutrient broth (NB), applied either undiluted or diluted 2, 4, 10 and 20 folds. WFW and NB in the same volume and dilutions as the treatments were used as controls, as well as tap water. The Petri dishes were sealed with parafilm to prevent moisture loss and incubated in the dark for the first 48 h at 25 °C, followed by incubation under ambient light with natural day-night cycles for the next 5 days at the same temperature. Each day, the germination percentage (GP, %) for each treatment and control was determined by counting the number of germinated and non-germinated seeds. The minimal root length of 1 mm was used for evaluation of germination test as positive. These data were also used to calculate the mean germination time (MGT, days; Equation (5)), where n_i represents the number of seeds germinated on day d_i, and N is the total number of seeds germinated after 7 days. Additionally, the germination index (GI, seeds/day; Equation (6)) was calculated starting from day 3 of the germination period. At the end of the 7-day germination period, the following seedling parameters were measured: fresh mass (FM, g), dry mass (DM, g; after drying at 80 °C to constant mass), total length (TL, cm), root length (RL, cm), and shoot length (SL, cm). Based on these measurements, seedling vigour indices I and II (SVI-I and SVI-II; Equations (7) and (8), respectively [38]) were calculated.

$$\mathrm{M}\mathrm{G}\mathrm{T}={\displaystyle \frac{\sum ({\mathrm{n}}_{\mathrm{i}}\times {\mathrm{d}}_{\mathrm{i}})}{\mathrm{N}}}$$

(5)

$$\mathrm{G}\mathrm{I}=\sum \left({\displaystyle \frac{{\mathrm{n}}_3}{{\mathrm{d}}_3}}\right)+\dots \left({\displaystyle \frac{{\mathrm{n}}_7}{{\mathrm{d}}_7}}\right)$$

(6)

$$\mathrm{S}\mathrm{V}\mathrm{I}-\mathrm{I}=\mathrm{G}\mathrm{P}\times \mathrm{T}\mathrm{L}$$

(7)

$$\mathrm{S}\mathrm{V}\mathrm{I}-\mathrm{I}\mathrm{I}=\mathrm{G}\mathrm{P}\times \mathrm{D}\mathrm{M}$$

(8)

2.11. Statistical Analyses

The experimental data were analyzed using TIBCO Statistica^® 14.1.0 software (TIBCO Software Inc., part of Cloud Software Group, San Ramon, CA, USA) to evaluate statistical differences using the One-Way ANOVA, followed by Duncan’s multiple range post hoc test, taking into account 95% confidence intervals.

3. Results

3.1. Identification of the Producing Microorganism

The identification of producing microorganism was performed using 16S rRNA gene sequencing. The results showed the highest similarity of the producing microorganism with Bacillus mojavensis and Bacillus halotolerans based on BLAST analysis, with sequence similarity values around 98%. Phylogenetic analysis based on 16S rRNA sequences was performed to determine the relationship of the isolate with the closely related Bacillus species. The constructed phylogenetic tree (Figure 1) confirmed the BLAST results, clearly showing the closest relationship of the isolate with Bacillus mojavensis and Bacillus halotolerans strains. The 16S rRNA sequence of Bacillus sp. 10/R has been deposited in the GenBank database under the accession number PX210502.

3.2. Biochemical Profiling of the Strain Bacillus sp. 10/R

In order to investigate the biochemical characteristics of the Bacillus sp. 10/R isolate, API test kits were used. The results obtained after 48 h of incubation of the inoculated API 50 CH strips, which include 50 biochemical tests for the study of bacterial carbohydrate metabolism, are shown in

Table 1. The results of API 50 CH biochemical tests for the isolate Bacillus sp. 10/R.

API 50 CH Test Strip Results
Glycerol	+	D-mannitol	+	D-raffinose	+
Erythritol	−	D-sorbitol	+	Starch (amidon)	+
D-arabinose	−	methyl-α-D-mannopyranoside	−	Glycogen	+
L-arabinose	+	methyl- α-D-glycopyranoside	+	Xylitol	−
D-ribose	+	N-acetylglucosamide	−	Gentiobiose	−
D-xylose	+	Amygdalin	+	D-turanose	−
L-xylose	−	Arbutin	+	L-lyxose	−
D-adonitol	−	Esculin	+	D-tagatose	+
Methyl-β-D-xylopyranoside	−	Salicin	+	D-fucose	−
D-galactose	−	D-cellobiose	+	L-fucose	−
D-glucose	+	D-maltose	+	D-arabitol	−
D-fructose	+	D-lactose	−	L-arabitol	−
D-mannose	+	D-melibiose	−	Potassium gluconate	+
L-sorbose	−	D-saccharose	+	Potassium 2-ketoglyconate	−
L-rhamnose	−	D-trehalose	+	Potassium 5-ketoglyconate	−
Dulcitol	−	Inulin	+
Inositol	−	D-melezitose	−

“+” designates positive rection results, while “–“ designates negative reaction result.

.

The second API kit used for the biochemical characterization of Bacillus sp. 10/R was API 20 E. Since the last 10 tests included in this kit are already covered by the API 50 CH test strip,

Table 2. The results of API 20 E biochemical tests for the isolate Bacillus sp. 10/R.

API 20 E Test Strip Results
ONPG	+	ADH	+	LDC	−	ODC	−	CIT	+
H2S	−	URE	−	TDA	−	IND	+	VP	+

ONPG–β-galactosidase; ADH–arginine dihydrolase; LDC–lysine decarboxylase; ODC–ornithine decarboxylase; CIT–citrate utilization; H₂S–H₂S production; URE–urease; TDA–tryptophane deaminase; IND–indole production; VP–acetoin production; “+” designates positive rection results, while “–“ designates negative reaction result.

shows the results of the remaining 10 tests from the API 20E kit.

The results of the API ZYM tests are presented in

Table 3. The results of API ZYM biochemical tests for the isolate Bacillus sp. 10/R.

API ZYM Test Strip Results
Control		Acid phosphatase	+
Alkaline phosphatase	+	Naphthol-AS-BI-phosphohydrolase	+
Esterase (C 4)	+	α-galactosidase	−
Esterase Lipase (C 8)	+	β-galactosidase	−
Lipase (C 14)	−	β-glucuronidase	−
Leucine arylamidase	+	α-glucosidase	+
Valine arylamidase	+	β-glucosidase	−
Cystine arylamidase	−	N-acetyl-β-glucosaminidase	−
Trypsin	−	α-mannosidase	−
α-chymotrypsin	−	α-fucosidase	−

“+” designates positive rection results, while “–“ designates negative reaction result.

. API ZYM is a semi-quantitative micromethod used for the investigation of enzymatic activities.

3.3. Plant Growth Promoting Traits of the Strain Bacillus sp. 10/R

In order to assess the potential of the investigated Bacillus isolate as a bioinoculant for plants, several traits considered crucial for plant growth promotion (PGP) were evaluated. These included enzymatic activity of the hydrolytic enzymes (cellulase, xylanase, pectinase and protease), indole-3-acetic acid (IAA) production, phosphate solubilization, and ACC deaminase production. The results of the enzymatic activity assays are shown in Figure 2, presenting growth and halo zone diameters, as well as the enzymatic activity index (EAI). The lowest EAI value was recorded for protease activity, where no halo zone appeared around the bacterial growth. On the other hand, the highest EAI was observed for pectinase production, with a value of 2.79, indicating a strong capacity for the extracellular production of this enzyme. The enzymatic activity indices for cellulase and xylanase were 2.33 and 1.75, respectively.

The ability of the Bacillus sp. 10/R isolate to produce ACC deaminase was confirmed by its growth on DF minimal medium supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole nitrogen source. A growth zone of 54.67 ± 0.58 mm was observed, indicating the strain’s capability to utilize ACC. In contrast, phosphate solubilization activity was relatively low, with a phosphate solubilization index (PSI) of only 1.13 ± 0.07. The results of these assays are presented in

Table 4. Plant growth promoting traits of Bacillus sp. 10/R in terms of ACC deaminase production and phosphate solubilization.

Parameter	Growth Zone Diameter (mm)	Halo Zone Diameter (mm)	PSI
ACC deaminase production	54.67 ± 0.58	/	/
Phosphate solubilization	11.17 ± 0.29	12.67 ± 1.15	1.13 ± 0.07

PSI—phosphate solubilization index, ACC—1-aminocyclopropane-1-carboxylic acid.

.

The ability of Bacillus sp. 10/R to produce indole-3-acetic acid (IAA), a key phytohormone involved in the regulation of plant growth and root development, was evaluated using a colorimetric method based on reaction with Salkowski’s reagent. The assay was performed using the cultivation broth supernatant after incubation under the optimized conditions. The concentration of IAA detected in the supernatant was 9.66 µg/mL, indicating a moderate level of auxin production by the isolate Bacillus sp. 10/R.

3.4. Basic Nutritional Profile of WFW Medium and Cultivation Broth

The characterization of the WFW medium was performed by analyzing total and soluble solids, sugar content, protein, cellulose, and alcohol content in order to assess its potential as a substrate for microbial bioconversion. The same parameters were evaluated after 48 h of Bacillus sp. 10/R cultivation to determine changes in the nutritional profile caused by bacterial activity. The obtained results are presented in

Table 5. The basic nutritional and pH value profile of WFW medium before and after cultivation.

Parameter	Before Cultivation	After Cultivation
Total solids (%)	7.96 ± 0.06	6.99 ± 0.13
Soluble solids (°Bx)	6.42 ± 0.35	6.00 ± 0.00
Total sugar content (%)	6.61 ± 1.35	2.68 ± 0.06
Reducing sugar content (%)	4.28 ± 0.47	/
Protein content (%)	0.64 ± 0.06	0.29 ± 0.02
Cellulose content (%)	0.01 ± 0.01	0.00 ± 0.00
pH value	3.54 ± 0.27	5.95 ± 0.25

.

3.5. Bacterial Growth in WFW Medium During 96 h of Cultivation

To investigate bacterial survival and growth in the used winery waste-based medium, biomass content of Bacillus sp. 10/R was measured every 24 h of cultivation. The drop plate method was employed for biomass determination, and the obtained results are presented in Figure 3. The biomass increased by approximately 2 log units after 48 h of cultivation and remained close to that level over the following 48 h. Considering that the maximum bacterial content was observed at 48 h, this time point was selected as the final cultivation time for subsequent analyses.

3.6. Enzyme Activity in Liquid WFW-Based Culture

Throughout the 48 h-cultivation of Bacillus sp. 10/R on WFW-based medium, enzymatic activity was measured by the DNS method for pectinase activity, while cellulase and xylanase activity was determined using CellG5 method and XylX6 method using the commercial enzyme assay kits. The obtained results in terms of enzymatic activity are presented in

Table 6. Pectinase, cellulase, and xylanase activities of Bacillus sp. 10/R cultivated using the winery flotation wastewater medium, monitored over 48 h of cultivation.

Time of Cultivation (h)	Pectinase Activity (U/mL)	Cellulase Activity (CellG5 U/mL)	Xylanase Activity (XylX6 U/mL)
12	2.19	0.00	0.00
24	2.19	0.01	0.00
36	2.19	0.00	0.00
48	2.20	0.08	0.00

U/mL = (µMol/(mL × min)).

.

3.7. Seed Germination

The efficiency of Bacillus sp. 10/R cultivation broth in promoting barley seed germination was evaluated using the cultivation broths prepared on WFW and nutrient broth (NB) at different dilutions. These treatments were compared with the corresponding dilutions of WFW and NB alone, as well as with tap water, which served as the negative control. Germination rate was recorded daily during a 7-day experiment. After 7 days, root and shoot lengths of each seedling were measured, together with fresh mass. Dry mass was determined after drying at 80 °C to constant weight. The results for germination rate, seedling length, and fresh and dry mass are presented in Figure 4. The experimental data obtained after 7 days were further used to calculate mean germination time (MGT), germination index (GI), and seedling vigor indices I and II (SVI-I and SVI-II), as shown in Figure 5. In both figures, cultivation broths are designated as CB.

As illustrated in Figure 4 and Figure 5, the concentration of cultivation broths and media strongly influenced germination rate as well as seedling length and biomass. The WFW-based cultivation broth applied at 10% concentration had a particularly pronounced effect on shoot elongation, resulting in the highest total seedling length (98.4 mm) among all treatments. The highest germination percentage (60%) was recorded for seeds treated with 10% WFW-CB and 50% WFW, whereas no germination occurred in treatments with 100% WFW and 100% WFW-CB. Regarding the plant biomass synthesis, fresh mass was highest in seeds treated with 10% WFW-CB, while dry mass was greatest in seedlings treated with 10% NB-CB. Overall, all treatments with cultivation broths yielded higher dry mass compared to media alone and the tap water control.

For MGT (Figure 5), no values could be determined for seeds treated with 100% WFW-CB and 100% WFW due to the absence of germination. The highest GI value was observed in seeds treated with 10% WFW-CB (1.84 seeds/day), followed closely by 50% WFW (1.83 seeds/day). Among the treatments where germination occurred, the lowest GI values were recorded for 100% NB-CB (0.17), 50% WFW-CB (0.20), and 50% NB-CB (0.20).

4. Discussion

In recent years, microorganisms used as bioinoculants and PGP microorganisms have garnered increasing attention due to their potential to enhance sustainable agriculture. Their ability to stimulate plant growth and improve plant health is attributed to a variety of direct and indirect mechanisms [39,40,41,42]. These include direct PGP mechanism facilitating nutrient acquisition and phytohormones production (auxins, gibberellins, abscisic acid, and cytokinins), as well indirect PGP mechanisms targeting biocontrol of competing organisms and plant pathogens, as well as production of compounds involved in quorum sensing and plant systemic resistance induction [43,44]. The most commonly studied and applied PGP microorganisms include species from the genera Bacillus, Pseudomonas, Azospirillum, Azotobacter, and Rhizobium, as well as beneficial fungi such as Trichoderma [45,46]. The genus Bacillus stands out as one of the most promising bacterial genera due to its wide range of beneficial activities and is among the most commonly used for plant growth promotion. Numerous species within this genus, such as B. velezensis, B. subtilis, B. amyloliquefaciens, B. macerans, B. circulans, B. azotofixans, and B. coagulans, have been well-documented for their ability to enhance plant growth through various mechanisms [47,48].

4.1. PGP Traits of B. mojavensis/B. halotolerans Strains

The grapevine rhizosphere isolate Bacillus sp. 10/R used in this study was molecularly identified using 16S rRNA gene sequencing and showed the highest similarity with Bacillus mojavensis and Bacillus halotolerans, with sequence identity values around 98%. Although this level of similarity suggests a close relationship with these species, it does not reach the ≥98.65% threshold generally accepted for confident species-level identification based on 16S rRNA. Therefore, the isolate was designated as Bacillus sp., most closely related to the B. mojavensis/B. halotolerans clade. To achieve precise taxonomic resolution, further studies such as multilocus sequence analysis or whole-genome sequencing would be required. Previous studies have highlighted the potential of B. mojavensis as a PGP species with multiple beneficial traits. For instance, Prajakta et al. (2019) [49] demonstrated that B. mojavensis produces volatile metabolites with antifungal activity against Rhizoctonia solani, along with siderophores, IAA, chitinases, and phosphate-solubilizing ability, confirming its dual role in biocontrol and plant growth promotion. Ghazala et al. (2023) [50] showed that B. mojavensis strain PGPB I4 enhanced wheat growth under salinity stress by improving physiological parameters, chlorophyll content, and antioxidant activity, whereas Sdiri et al. (2024) [51] reported that B. mojavensis I4 (BmI4) improved potato growth and tolerance to salinity through similar mechanisms, including enhanced membrane stability and modulation of antioxidant enzymes. In addition, Danish et al. (2022) [52] showed that B. mojavensis BZ-13 alleviated silver nanoparticle-induced toxicity in Withania somnifera, improving plant biomass, photosynthetic efficiency, and secondary metabolite accumulation while reducing oxidative stress and nanoparticle uptake. Several studies have highlighted the potential of Bacillus halotolerans as a PGP agent under salinity stress. Kapadla et al. (2022) [53] reported that the strain AD9, isolated from the saline coastal soil, exhibited multiple PGP traits at high salt concentrations (15–20%), including high levels of ammonia production and phosphate solubilization. Inoculation with AD9 significantly improved rice growth under salinity stress, resulting in increased root length, plant dry weight, and tiller number compared to the control. Similarly, Oliva et al. (2023) [54] characterized halotolerant strains including Bacillus sp. M21 and M23, closely related to B. halotolerans, and demonstrated that their PGP activities, such as IAA production and ammonium release, were strongly influenced by the present NaCl levels.

4.2. Biochemical Profile of Bacillus sp. 10/R

The biochemical characterization of Bacillus sp. 10/R using API 50 CH, API 20 E, and API ZYM systems revealed a versatile metabolic profile. The isolate was capable of fermenting a broad range of carbohydrates, including glucose, maltose, arabinose, salicin, cellobiose, and trehalose, indicating strong saccharolytic potential, while failing to utilize sugars such as galactose, turanose, lyxose, and dulcitol. API 20 E results showed positive reactions for β-galactosidase, arginine dihydrolase, citrate utilization, indole production and acetoine production, demonstrating the strain’s ability to metabolize diverse substrates and produce key metabolic intermediates, consistent with other Bacillus species. The enzymatic profiling using API ZYM indicated high activity of esterases (C4 and C8), alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, reflecting the strain’s potential to degrade a variety of substrates, including proteins and phosphoesters. In contrast, activities of lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, β-glucosidase, and other glycoside hydrolases were absent or weak, suggesting selective enzymatic capabilities. Overall, these results demonstrate that Bacillus sp. 10/R possesses a diverse enzymatic repertoire and carbohydrate utilization profile, which may contribute to its adaptability and potential for biotechnological applications such as plant growth promotion and biocontrol.

4.3. Enzymatic Activity of Bacillus sp. 10/R

The remarkable adaptability of Bacillus strains to diverse environmental conditions is one of the key advantages of this genus, enabling cultivation on a wide range of substrates, including nutrient-rich industrial waste streams [55]. The production of hydrolytic enzymes provides these bacteria with the ability to access and utilize nutrients from complex substrates. Screening for hydrolytic enzyme activity among Bacillus isolates is therefore a common step in the assessment of their PGP potential, since such enzymatic activity not only enhances the utilization of nutrients available in the soil by plants and other beneficial soil microorganisms, but also contributes to the improved degradation of crop residues and direct antagonism against phytopathogens through cell wall degradation mediated by these enzymes [9]. Cellulase, xylanase, pectinase, and protease activities were evaluated as the key enzymatic functions involved in degradation of the predominant organic substrates typically present in soil. Cellulases target cellulose originating from plant residues, xylanases hydrolyze hemicellulose components such as xylan from lignocellulosic material, pectinases degrade pectin-rich components derived from fruit and vegetable tissues, while proteases break down proteins released from microbial biomass and other organic inputs. The selection of these enzymes was based on their central role in mineralizing the major organic fractions in soil and facilitating nutrient cycling [56,57].

Using a semi-quantitative agar plate assay, enzymatic activity was expressed as the enzymatic activity index (EAI) to provide a comparative measure of hydrolytic potential, based on the extracellular enzymatic activity, as shown in Figure 1. The investigated isolate Bacillus sp. 10/R exhibited the highest extracellular pectinase activity (EAI = 2.79), indicating strong capacity for hydrolyzing pectin-rich substrates and suggesting adaptation to environments enriched with fruit- and vegetable-derived residues, which is also beneficial in case of using grape-based winery effluents for cultivation of the aforementioned strain, as in this study. According to Kabir and Tasmim (2019) [58], a halo zone diameter of ≥ 15 mm indicates very good pectinolytic activity, which is consistent with the results obtained in our study. Among 95 isolates from coffee pulp investigated by Oumer and Abate (2018) [59], 31.58% exhibited pectinolytic activity as evidenced by clear zones around bacterial growth, while 10 isolates were considered good pectinase producers with an EAI ≥ 2.00. Cellulase activity (EAI = 2.33) was also notable, reflecting the potential to degrade cellulose-containing plant material, whereas xylanase activity was comparatively lower (EAI = 1.75), which may indicate a limited hemicellulolytic capacity under the applied experimental conditions. In a study by Castaldi et al. (2021) [60], among 22 Bacillus isolates, the highest proportion of strains produced xylanase and protease activities (90%), while 80% exhibited cellulolytic activity. Furthermore, cellulase activity was investigated among 398 isolates from various environments and detected in 6.5% of them, with enzymatic indexes ranging between 0.34 and 5.2 [61]. Xylanase EAI achieved by Bacillus sp. BioSol021 was 2.85, which is significantly higher than the result achieved by Bacillus sp. 10/R [9]. In contrast, protease activity showed an EAI of 1.00, as no clear hydrolysis zone surrounding the growth area was observed. Notably, the largest growth zone (21.67 ± 0.58 mm) was recorded when the isolate was cultivated on a protein-based medium supplemented with skim milk as the nutrient source. Although no surrounding clear zone of extracellular protein hydrolysis was detected, the pronounced colony growth suggests that the isolate was able to effectively utilize the available amino acid-based nutrients subsequently with their enzymatic release from milk proteins. This may imply the presence of cell-bound or intracellular proteolytic enzymes, the uptake of small peptides and amino acids naturally present in skim milk, or an alternative metabolic adaptation that supports growth without extensive extracellular protein degradation.

4.4. Direct and Indirect PGP Mechanisms Exhibited by Bacillus sp. 10/R

Abiotic and biotic stress significantly reduce crop growth and productivity by altering plant morphological and physiological traits, often through increased ethylene production, which inhibits root development. Plant growth-promoting microorganisms can alleviate these negative effects, with ACC deaminase production representing a key mechanism of stress mitigation. This enzyme hydrolyzes ACC, the ethylene precursor, into ammonia and α-ketobutyrate, thereby reducing ethylene levels under stress conditions and promoting root growth and overall plant development [62,63]. Diverse microbial taxa, including Acinetobacter, Aeromonas, Burkholderia, Bacillus, Enterobacter, Hallobacillus, Sphingomonas and Streptomyces, have been identified as efficient ACC deaminase producers. Exploiting such microbes offers a promising strategy to improve plant tolerance to environmental stresses, including drought [64]. In the present study, the ability of Bacillus sp. 10/R to grow on ACC-supplemented medium devoid of other nitrogen sources, reaching a colony diameter of 54.67 ± 0.58 mm, indicates substantial ACC deaminase activity, further supporting its potential role in enhancing plant stress tolerance. After nitrogen, phosphorus represents the second most essential macronutrient required for optimal plant growth and development [65]. Phosphorus is essential for crop development, particularly during early vegetative growth, seedling establishment, and maturation, where it plays a key role in enhancing seed quality and yield through its involvement in various morphological and physiological processes. Phosphate-solubilizing microorganisms facilitate the release of phosphorus from both inorganic and organic soil pools via solubilization and mineralization, thereby acting as PGP agents. This activity not only improves plant growth and grain yield but also reduces the need for chemical fertilizers [66]. Candales et al. (2017) [67] investigated the PGP potential of Bacillus isolates and found that, among 15 tested strains, only two, Bacillus subtilis GIBI 200 and Bacillus pumilus GIBI 206, were able to solubilize phosphorus. In the study by Kumari et al. (2024) [68], five out of seven bacterial isolates demonstrated phosphate solubilization on Pikovskaya medium, with PSI values ranging from 2.3 to 4.5. Similarly, PGP Bacillus strains examined by Chandra et al. (2021) [69] exhibited PSI values between 2.5 and 3.6. In contrast, the results obtained in our study indicate a low phosphate-solubilizing ability of Bacillus sp. 10/R compared to the aforementioned studies, with over 50% lower PSI values (1.13 ± 0.07).

IAA is primarily responsible for root elongation by stimulating cell division and tissue differentiation, which subsequently increases plant fresh mass. Therefore, its production is considered one of the main PGP traits to be assessed when evaluating the potential active components of biostimulants [70]. The production of IAA as a plant growth-promoting trait has been widely investigated in numerous studies, showing variable results. Goud et al. (2025) [71] reported that, out of 80 bacterial isolates, 67 were capable of producing IAA and its derivatives, indicating that IAA production is a common feature among many bacteria. In their study, IAA was synthesized predominantly via the tryptophan-dependent pathway, with concentrations ranging from 3.8 to 70.0 µg/mL. Vlajkov et al. (2023) [9] reported an IAA concentration of 15 µg/mL for Bacillus sp. BioSol021, while in the study by Chandra et al. (2021) [69], IAA concentrations ranged from 2.49 to 5.47 µg/mL, with the highest level produced by Bacillus subtilis strain GIBI 200. In the present study, Bacillus sp. 10/R produced IAA at a concentration of 9.66 µg/mL, which demonstrates a substantial capacity for IAA synthesis.

4.5. Winery Flotation Wastewater as a Substrate for Bacillus sp. 10/R Growth

Winery wastewater (WWW) represents a major effluent stream of the winemaking industry, generated mainly from cleaning and processing operations, and is characterized by high volumes, strong seasonal variability, and an acidic pH value. Its composition includes elevated concentrations of readily biodegradable organic matter (sugars, ethanol, organic acids), suspended solids, and polyphenolic compounds, alongside moderate levels of nitrogen, phosphorus, and salts [72,73]. The reported COD (chemical oxygen demand) values typically range between 320 and 49,000 mg/L, while BOD₅ (biological oxygen demand) averages around 6500 mg/L, highlighting its high organic strength and biodegradability [73], However, the low N and P relative to organic load often necessitates nutrient supplementation for microbial processes. Despite these challenges, the nutrient-rich nature of WWW and its biodegradability make it a suitable substrate for microbial bioconversion processes, including anaerobic digestion, where it can serve as a source for bioenergy and value-added products such as biofertilizers [72,74].

To assess the potential of WFW as a substrate for the cultivation of Bacillus sp. 10/R, its basic nutritional profile was investigated, including total and soluble solids, total and reducing sugar content, protein content, and alcohol content. The same analyses were performed at the end of the cultivation period to monitor nutrient consumption by the bacteria. The results presented in Table 5 correspond to WFW diluted three times, which was the form used for the cultivation of Bacillus sp. 10/R. The initial pH value of the prepared medium was 3.54 ± 0.27, representing an acidic environment, and was subsequently adjusted to 6.5 ± 0.2 prior to inoculation. It has been gradually dropping to 5.95 ± 0.25 by the end of the cultivation, thus suggesting the production of acidic bacterial metabolites during the cultivation course. The cultivation was monitored over time, and although samples were collected beyond 48 h, this time point was chosen as the endpoint for compositional analyses, as it corresponded to the maximum biomass content and peak viable cell density. Prior to inoculation, the medium contained 7.96 ± 0.06% total solids and 6.42 ± 0.35 °Bx soluble solids, reflecting a high load of dissolved organic matter. The total sugar content was 6.61 ± 1.35%, of which 4.28 ± 0.47% were reducing sugars, indicating a substantial proportion of readily utilizable monosaccharides and disaccharides. The protein content of 0.64 ± 0.06% suggested the presence of organic nitrogen sources, while cellulose content was negligible (0.01 ± 0.01%), as expected for a liquid waste stream derived from fruit processing, considering that the major part of insoluble carbohydrates remains in the solid fraction after grape processing [21]. After 48 h of cultivation, total solids decreased to 6.99 ± 0.13%, and soluble solids to 6.00 ± 0.00 °Bx. Total sugars declined to 2.68 ± 0.06%, whereas reducing sugars were completely depleted, indicating the rapid utilisation of simple carbohydrates by the bacteria. Protein content dropped to 0.29 ± 0.02%, corresponding to a ~55% reduction, suggesting significant assimilation of organic nitrogen during growth. Cellulose was undetectable at the end of cultivation. The results demonstrate that the medium initially provided sufficient amounts of readily available carbon and nitrogen sources to support Bacillus sp. 10/R growth, but that growth cessation was likely associated with the depletion of utilizable reducing sugars and organic nitrogen, rather than the exhaustion of total carbohydrate content.

Enzyme activity of Bacillus sp. 10/R cultivated on WFW medium was monitored over 48 h, at four time points (12, 24, 36 and 48 h). Pectinase activity was determined using the DNS method, while cellulase and xylanase activities were assessed using the CellG5 and XylX6 commercial assay kits, respectively. The culture supernatants were obtained by centrifugation of cultivation broths at 10,000 rpm for 10 min and used for enzyme activity measurements. Pectinase activity remained relatively constant during cultivation, with values of 2.19 U/mL at the first three time points and 2.20 U/mL at 48 h. In the study by Mohandas et al. (2018) [27], using the same method to assess pectinase activity of Bacillus sonorensis MPTD1, maximum activity reached 2.43 (μM/mL)/min. Among four soil-derived Bacillus isolates (B. firmus, B. coagulans, B. endophyticus and B. vietnamensis), B. firmus exhibited the highest pectinase activity, recorded as 0.0053 IU/mL/min [75]. Utami et al. (2022) [76] reported that pectinase activity of Bacillus sp. 2P11, monitored over 96 h, peaked at 72 h with a value of 0.391 U/mL, suggesting the strong pectinolytic activity of the strain Bacillus sp. 10/R used in this study.

In contrast to pectinase activity, no xylanase activity was detected under the tested conditions, while cellulase activity was low, with values of 0.01 and 0.08 CellG5 U/mL measured at 24 h and 48 h, respectively. The absence of detectable cellulase and xylanase activities in the supernatants of Bacillus sp. 10/R cultivated on WFW medium can be explained by the composition of the substrate. Previous analysis of WFW showed that the cellulose content was extremely low (0.01%, calculated value), which likely limited induction of cellulolytic activity. Since xylan is typically present in close association with cellulose in lignocellulosic materials, its concentration in WFW can also be assumed to be negligible. Given that production of hydrolytic enzymes such as cellulases and xylanases in Bacillus spp. is strongly regulated by substrate availability and is usually induced only in the presence of polysaccharides or their degradation products, the absence of sufficient inducers in WFW likely resulted in the lack of detectable enzyme activity under the tested conditions [77,78,79,80]. The substrate dependence of enzyme synthesis was further confirmed in this study by agar plate assays, where Bacillus sp. 10/R exhibited clear cellulase and xylanase activity on media supplemented with CMC and xylan, respectively. These findings indicate that the lack of activity in liquid cultures with WFW was not due to the inability of the strain to produce these enzymes, but rather to the absence of appropriate inducers.

4.6. Seed Treatment of Barley Using Circular Biostimulant Based on Bacillus sp. 10/R and Winery Flotation Wastewater

This study has investigated the effects of a seed treatment based on the plant biostimulant produced using Bacillus sp. 10/R and WFW on barley seed germination and the initial seedling growth. A wide range of studies have demonstrated that Bacillus isolates can significantly enhance seed germination and early seedling growth in various plant species [67,81,82,83,84]. Among them there are some investigations about Bacillus beneficial effects on barley seeds. For instance, specific Bacillus strains, such as BMG1 and BMG2, have been shown to increase the germination percentage of barley seeds and promote substantial elongation of both roots and shoots. The isolate BMG1 was found to be exceptionally effective, increasing the shoot length of barley by 2.23 times compared to the untreated control, while the isolate PAZE-6 demonstrated the most significant effect on root growth, increasing the root length of barley by up to 60% [85]. Moreover, research by Teker Yıldız and Acar (2025) [86] indicates that Bacillus strains can alleviate abiotic stresses, with Bacillus thuringiensis and Bacillus cereus helping to improve germination and growth under saline conditions The inoculation of Bacillus cereus led to a 17% increase in root length in barley seedlings compared to stressed but untreated plants. Bacillus thuringiensis was even more effective at increasing root length [80]. Co-inoculated phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis) and nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum) resulted in a grain yield of up to 4.5 t/ha (tons per hectare) in spring barley. This was a significant increase compared to the control, which produced 3.37 t/ha [87]. The effect of Bacillus sp. 10/R cultivation broths on barley germination revealed a clear dependence on both the type of medium and the applied concentration. As shown in Figure 4, undiluted WFW and WFW-CB completely inhibited seed germination, which points to the presence of inhibitory compounds in winery wastewater that are not sufficiently neutralized or metabolized even after microbial growth. Such effects are frequently reported for wastewater rich in polyphenols and other secondary metabolites that may exhibit phytotoxic properties at high concentrations [88]. In the study by Mosse et al. (2010) [88], the effects of undiluted winery wastewater on the germination and early growth of several crop species, including lucerne, millet, phalaris and barley were assessed, and it was demonstrated that high concentrations of polyphenols and other secondary metabolites in the wastewater completely inhibited seed germination and reduced seedling growth. However, partial dilution of the media drastically altered their impact in our study. The highest germination rate, reaching 60%, was observed with 10% WFW-CB and 50% WFW, suggesting that at sub-inhibitory concentrations certain components of WFW, together with microbial metabolites, may act as stimulants of germination. This dual effect indicates that WFW serves not only as a growth substrate for bacteria but also as a potential source of bioactive molecules, which at appropriate concentrations can enhance early plant development. With respect to biomass, fresh mass was highest in seedlings treated with 10% WFW-CB, while for dry mass the greatest value was recorded with undiluted NB-CB, followed by 10% WFW-CB. Importantly, in all cases cultivation broths promoted higher dry mass compared to the negative control (tap water), confirming that bacterial metabolites contributed positively to seedling development, likely through the combined action of hydrolytic enzymes, phytohormone-like compounds, and nutrient mobilization. In summary, the increase in the investigated barley germination and growth parameters indicated suitability of 10% WFW-CB as a potential plant biostimulant, as well as suitability of Bacillus sp. 10/R as a biostimulant active component, with a necessity to further investigate possible mechanisms of barley growth promotion in more detail taking into account plant responses and large scale in vivo trials.

5. Conclusions

The results presented in this study reveal that the isolate Bacillus sp. 10/R from the grapevine rhizosphere, taxonomically closely related to Bacillus mojavensis and Bacillus halotolerans, could be considered as a potential plant growth promoting agent, with proven abilities to synthesize hydrolytic enzymes facilitating plant and soil microflora nutrition, compounds modulating plant immune responses and plant growth hormone auxin. Furthermore, an environment-friendly proof of concept was reported when it comes to the biotechnological production process for obtaining a circular microbial biostimulant. In this study, we tested barley seed treatment, which was based on the aforementioned bacterial strain used as an active component and its ability to effectively utilize nutrients present in winery flotation wastewater, whose suitability as a substrate for bacterial growth was also proven. Further research should precisely elucidate the plant responses regarding the suggested plant growth promotion mechanisms, taking into account wider range of crops, as well larger-scale testing in greenhouse and field trials. The need to investigate other possible application methods beyond seed treatment, as well as defining the optimal dosage of the biostimulant, considering the potential plant growth inhibitory effects observed in this study and the necessity to understand its effects to the plant–environment ecosystem, has also been foreseen. The proposed bioprocess solution should also be further analyzed for its techno-economical feasibility to determine the cost–benefit ratio for wineries as waste substrate generators, producers of microbial biostimulants and farmers as the end-users, thus developing both industrial and agricultural symbiosis for food production with reduced environmental footprint.