Production of a Microbial Biofilm and Its Application on Tomato Seeds to Improve Crop Development in a Lead-Contaminated Substrate

Abstract

The plant growth-promoting bacterium, B. subtilis subsp. Spizizizenii, has been proven to develop a biofilm under certain culture conditions, which can be applied as an efficient bioinoculant. Biofilm can be produced cost-effectively using biodiesel byproduct glycerol as a carbon source. Soils from urban peripheries may contain very high lead (Pb) levels. The main aim of this study was to assess the impact of biofilm seed inoculation on plant development and fruit quality of tomatoes growing on a Pb-contaminated substrate. Also, effects of excess Pb on biofilm production, stability, and seed germination were analyzed. B. subtilis biofilm was produced with Pb concentrations ranging from 0 to 300 ppm. Biofilm stability was tested at 4 °C and 25 °C. The impacts of Pb and inoculation on seed germination were evaluated in laboratory conditions, while the impacts on plant agronomic parameters were assessed via a greenhouse assay. Adding Pb to the culture medium increased biofilm production by about 20%. Regardless of Pb level, biofilms were more stable at 4 °C than at 25 °C. Beneficial effects of biofilm on germination were greater on seeds exposed to 200 and 300 ppm Pb. Excess Pb significantly reduced plant biomass and tomato yield. However, biofilm inoculation significantly increased plant aboveground and root biomass, plant height, leaf area, fruit number, and fruit size, regardless of substrate Pb excess. Tomato fruits of plants grown in the metal-contaminated substrate showed no significant increases in Pb concentration with respect to the control. In summary, the biofilm produced by B. subtilis subsp. spizizenii proved to be an effective bioinoculant to counteract the negative effects of substrate excess Pb on tomato germination, growth, and production.

1. Introduction

In the absence of contamination, soil heavy metal concentration merely depends on the composition of the parent material and the soil genetic processes affecting weathering and element speciation. However, human activities, especially through industry, mining and metal smelting processes, releases considerable amounts of heavy metals into the environment, which is the most frequent cause of high concentrations reaching toxic levels [1,2,3]. Higher Pb concentrations have been reported in soils located near industrial facilities, roads, ammunition depots, sport and military shooting ranges than in soils from more isolated areas [4]. Inputs of Pb in agricultural soils are due to the use of Pb arsenate-based pesticides, application of Pb-rich wastewater irrigation, inorganic phosphorus fertilizers and sewage sludge [5]. In particular, in urban and peri-urban soils, Pb has been ranked as the most frequent contaminant [6]. The different sources of external Pb additions can result in hotspots with toxic levels [5,6].

Lead (Pb) is one of the least mobile metals in soils and tends to remain localized at the topmost layer and does not leach into deeper horizons. Usually, it is retained in surface humus and accumulates in plant roots. This heavy metal is mainly found in soil as a cation in the Pb⁺² form, which can react to form PbS, a still higher insoluble compound. In addition, a significant Pb fraction is complexed with organic matter and can adsorb clay oxides and silicates. At slightly basic pH, Pb can precipitate as carbonate, hydroxide, or phosphate. In alkaline environments, it can be mobilized by forming complexes with organic matter and hydroxide ions. Due to analogous metallic properties and characteristics, Cd and Zn are also often found in Pb-contaminated soils [3,6,7].

Effects of this metal on plants are complex and mainly related to inhibition of germination, chlorosis, reduction of root length, and retardation of whole plant growth [3,8,9]. Also, photosynthetic pathways are negatively affected because the metal disorganizes the structure of chloroplasts and inhibits the production of essential pigments such as carotenoids, chlorophyll, and plastoquinone [9,10]. Furthermore, Pb can alter cell membrane permeability, inhibit enzymes possessing sulfhydryl groups, decrease water content, and cause an imbalance in mineral distribution [9,11]. Additionally, Pb toxicity has been shown to cause blockages in the electron transport chain and the Calvin cycle, as well as the closure of stomatal pores [12].

The negative impacts of Pb on crops, however, can be alleviated through the use of plant growth-promoting bacteria (PGPB). These microorganisms may have a highly varied metabolism, which is most dependent on the habitat in which they are found. Some of the mechanisms stimulating plant growth and protecting plants from abiotic stresses are related to the synthesis of indole acetic acid-type growth regulators and cytokines, nitrogen fixation, prevention of excessive ethylene secretion, production of siderophores, and antibiotic substances [13]. Relevant for these microorganisms is that their capacity to enhance plant growth depends not only on the bacteria itself but also on the plant that is colonized. Therefore, the same bacterium can be highly effective for one crop but not necessarily for another one [14]. Several PGPBs demonstrated a great potential to promote the growth of various plants in metal-contaminated environments. The most important bacteria strains used until now for the formulation of bioinoculants belong to the genus Bacillus [15,16]. They were used both for increasing plant productivity and also for phytoremediation purposes. Typically, Bacillus inoculants colonize the rhizosphere and also build up endophytic associations within numerous plant species [14]. Another important characteristic of these inoculants lies in the formation of endospores, which are resistance structures that allow the cells to maintain their viability under adverse environmental conditions. This is important in real-world applications because commercial inoculants can be stored for longer periods while retaining their viability.

Among the complex mechanisms employed by B. subtilis to mitigate toxic impacts of metals, the most common are biosorption processes [17,18,19,20]. Furthermore, as Gram-positive bacteria, this microorganism contains teichoic acids in the peptidoglycan layers that form their cell wall, which contribute to the retention of metal ions [21,22]. In particular, B. subtilis subsp. spizizenii was shown to have attributes of growth-promoting bacteria by increasing the growth of lettuce, tomato, and soybean. The mechanisms involved in this improvement included the production of phytohormones, the release of antifungal substances, and the solubilization of phosphates [23,24,25,26].

In laboratory conditions, B. subtilis can develop either as a free-living (planktonic) cell or produce biofilm [27]. The biofilm constitutes a highly structured community of bacteria surrounded by a matrix of extracellular polymeric substances (EPSs) [28,29]. Compared to their planktonic cell counterpart, biofilms offer multiple benefits, among others protection of cells against adverse environmental stresses (toxic chemicals, pH changes, dehydration, and predation) [30,31]. In addition, they enable cell-to-cell communication through the expression of quorum sensing molecules, facilitate the exchange of genetic material (such as horizontal gene transfer) and ensure nutrient availability as well as the persistence of various metabolic functions [29,32,33,34]. EPSs are high molecular weight biopolymers, including proteins, cellulose-rich polysaccharides, nucleic acids and lipids [34]. Several researchers have shown that the negatively charged functional groups and ligands of EPSs can act by capturing metal ions [19,35,36]. In addition, the enzymatic activity that develops in the EPS matrix plays a key role in the detoxification of metals, facilitating their transformation and subsequent precipitation in the polymer mass.

Tomato is one of the most important crops worldwide, for both its nutritional and economic value. It is a source of vitamins C, K, A, E, and B9, containing minerals such as K, Mg, P, Ca, and Fe; antioxidants, such as lycopene; and variable amounts of flavonoids [37,38]. Currently, tomato production faces numerous biotic and abiotic challenges [39]. To protect food safety and human health, national and international organizations have established maximum permissible levels of lead in food meant for human consumption. Examples of such regulation were provided by FAO/WHO [4], the US Environmental Protection Agency (EPA) [40], and the European Commission Regulations [41]. The maximum permissible Pb concentration for tomato fruits following these organizations is 0.1 mgkg⁻¹ wet weight. Previous research showed that the biofilm produced by Bacillus subtilis subsp. spizizenii is a better promoter of lettuce (Lactuca sativa L.) and tomato (Solanum lycopersicum L.) plant growth than the respective planktonic form [25,26]. This was attributed to the fact that the biofilm produced by this bacterium contains vegetative cells but also spores, and therefore it is more valuable in a real-world context because of a high stability and survival ability [25,26]. These studies focused on the production of biofilm and its application as a biopreparation to enhance seed germination and plant growth. However, the impact of B. subtilis biofilm as a natural agent to support plant growth under environmental stress conditions, such as heavy metal surplus, has been almost not addressed, and as stated previously, Pb contamination is widespread in urban peripheries. Therefore, the main objective of this study was to evaluate the impact of seed inoculation with biofilm produced by B. subtilis subsp. spizizenii on the growth and fruit quality of tomatoes grown on a Pb-contaminated substrate. Additionally, impacts of Pb concentration on biofilm yield cultures and on seed germination were evaluated.

2. Materials and Methods

2.1. Bacterial Culture to Obtain Planktonic Cells and Biofilm

The endophytic strain Bacillus subtilis subsp. spizizenii (accession No. 6633) supplied by American Type Culture Collection (ATCC) was used in our research. This strain was preserved at the campus of the Faculty of Agronomy, Buenos Aires University (FAUBA). The bacterium was initiated from the stock culture before it was used for experiments and grown for 24 h at 30 °C on nutritive agar medium (meat extract, 3 g; peptone, 5 g; agar, 25 g; and deionized H₂O up to 1 L).

Both planktonic cells and biofilm from B. subtilis were produced in a similar culture medium, specifically a liquid minimum salt medium (MSM) added together with 1% glycerol as a carbon source and 55 mM L-glutamic acid as a nitrogen source. The MSM composition was 1 g/L K₂HPO₄, 0.3 g/L KH₂PO₄, 0.5 g/L NH₄Cl, 0.1 g/L NH₄NO₃, 0.1 g/L Na₂SO₄, 0.01 g/L MgSO₄ 7H₂O, 1 mg/L MnSO₄ 4H₂O, 1mg/L FeSO₄7H₂O, 0.5 g/L CaCl₂, and 0.01 g/L EDTA in deionized water. MSM vas neutral, with pH = 7 [42]. In this work, planktonic cells were needed to build up B. subtilis growth curves, while biofilm was employed for laboratory and greenhouse experiments. Incubations were performed using 500 mL Erlenmeyer flasks with 150 mL of culture medium. Bacteria were grown for 96 h at 30 °C. Planktonic cell production requires incubation under agitation, which was performed at 150 rpm. However, to produce biofilm at the air-liquid interface, static conditions are indispensable. The biofilm was removed manually using a glass rod, to which it remained attached. Then, it was then transferred to sterile tubes and saved for subsequent assays.

2.2. Planktonic Cells and Biofilm Production with Increasing Pb Concentrations

2.2.1. Microbial Growth in the Planktonic State

B. subtilis grew under continuous stirring at 150 rpm as previously described with increased concentrations of Pb, namely 0 (control), 50, 100, 200, and 300 ppm in the medium to obtain planktonic state cells. Lead chloride (PbCl₂) was added to the medium as a source of Pb. Bacterial growth caused growth media to appear cloudy. Growth curves were measured through the detection of light scattered in absorbance at 610 nm (OD_610nm) using a spectrophotometer. Growth curves were built up from 8 sequential absorbance measurements were made from 0 to 96 h.

2.2.2. Biofilm Yield

Biofilm was obtained, also using culture broth with increasing Pb concentrations, from 0 ppm (control) to 300 ppm by incubation under static conditions, as previously described. Once manually extracted from the Erlenmeyer flask, the biofilm was dried to constant weight on filter paper at 40° C to calculate yield.

2.2.3. Biofilm Stability

Biofilm stability was measured over time using B. subtilis cultures grown in MSM with 0, 50, 100, 200, and 300 ppm Pb at room temperature (24 ± 2 °C). The biofilm produced was detached from the Erlenmeyer every 5 days and dried at 40 °C to a constant weight. Temperature effects on biofilms were measured from B. subtilis grown in MSM at either 24 ± 2 °C, room temperature, or 4 ± 1 °C fridge temperature; again, biofilms were taken off every 5 days and weighed and handled in the same way as before.

2.3. Experimental Site and Design and Plant Material

Laboratory and greenhouse experiments were carried out at the Faculty of Agronomy, University of Buenos Aires (FAUBA), Argentina (S 34°45′37″, W 60°31′03″. Laboratory tests include determinations of germination and root length percent, while greenhouse trials were set up to quantify plant growth and fruit production.

The laboratory tests and the greenhouse trials were conducted according to a completely randomized design. Five treatments were examined in the germination tests, which involved the examination of five treatments with 60 seeds per treatment. Concerning the greenhouse experiment, four treatments were studied, and ten seedlings per treatment were transplanted and allowed to grow. Three replicates per treatment were performed. In the next sections, a more detailed description of the treatments is given.

Seeds of the Río Grande variety of S. lycopersicum were used for germination tests and greenhouse trials. This tomato variety is commercially produced in Argentina. As a determinate variety, it has a relatively large growth cycle, so that flowering and ripening are set at the same time, but later than on indeterminate varieties.

2.4. Seed Germination Tests

Seed germination tests were performed with and without biofilm inoculation and at five different Pb concentrations, namely 0, 50, 100, 200, and 300 ppm, resulting in ten different treatments. To remove epiphytic microorganisms, the seeds were disinfected with 70% alcohol and washed three times with sterile, distilled water before performing the test. Seed inoculation was thoroughly mixed by shaking together tomato seeds and biofilm for 30 min. In this way, and due to its great adhesiveness, the biofilm remained attached to the seeds.

Sterile Petri dishes were used to perform germination tests. These included a layer of sterile cotton wool covered with Whatman Grade 3 filter paper, where seeds were placed. The test was conducted for the different treatments moistening the filter paper to encourage germination with 5 mL of sterile, distilled water (control) or Pb solutions with 50, 100, 200, and 300 ppm. This procedure was performed without and with seed inoculation, using 60 seeds per treatment. Then Petri dishes were left in a dark place for 10 days at 22 °C. A visible radicle of 2 mm length was the criterion for successful germination [43].

The relative germination percentage (RG) was calculated according to the following:

$$\mathrm{R}\mathrm{G}(\%)=\left({\displaystyle \frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s} \mathrm{i}\mathrm{n} \mathrm{a} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}}\right)\times 100$$

(1)

Thereafter the Petri dishes were placed under daylight conditions and seedlings continued growing for 5 days. Then, 15 days after the experiment was setup, seedling radicles were measured. Relative root elongation (RRE) and relative percentage germination index (GI), respectively, were calculated as follows:

$$\mathrm{R}\mathrm{R}\mathrm{E}(\%)=\left({\displaystyle \frac{\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \mathrm{i}\mathrm{n} \mathrm{a} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}}\right)\times 100$$

(2)

$$\mathrm{G}\mathrm{I}(\%)=\left({\displaystyle \frac{\mathrm{R}\mathrm{G}\times \mathrm{R}\mathrm{R}\mathrm{E}}{100}}\right)$$

(3)

The GI index includes features about the three main germination phases, i.e., seed imbibition, seedling emergence, and radicle elongation. These values of this index show an inverse correlation to toxicity levels, as shown in

Table 1. Correspondence between GI and toxicity level, according to Emino [44].

Germination Index (GI%)	Toxicity Level
<50%	High
50–80%	Moderate
>80%	No toxicity

[44,45]. Therefore, for increasing GI values toxicity levels are classified as high, moderate and no toxic. Once GI was calculated, the correspondence, shown in Table 1, was used to assess how toxic the presence of substrate Pb was for seed germination.

2.5. Tomato Cropping in Greenhouse Conditions

A greenhouse experiment was set up to analyze the impacts of excess substrate Pb and seed inoculation on plant growth and fruit quality. This trial was carried out under sunlight. The treatment setup included the following: (1) control, no seed inoculation, no excess Pb without inoculation, and Pb surplus; (2) I, seed inoculation, and no excess Pb; (3) Pb, no seed inoculation, and 300 ppm excess Pb; and (4) I + Pb, seed inoculation, and 300 ppm excess Pb.

2.5.1. Commercial Substrate Composition and Addition of Excess Pb

Tomato plants at the greenhouse were grown on a mixture of commercial substrate and commercial compost in a 3:1 ratio. The commercial substrate was characterized by a moisture content of 50% and a pH of 5.8 and exhibited 55% organic matter content, 45% ash content, and a carbon/nitrogen ratio of 30%. The contaminated substrate was prepared by adding up to 300 ppm Pb to the commercial substrate.

The main criterion to select a 300 ppm Pb as a level of contamination in the substrate was based on the results of biofilm germination tests (Section 3.4). In addition, permissible limits of Pb have been taken into account, even if these may differ widely between countries [4,6,46]. For example, in Argentina, soils with Pb concentrations lower than 275 ppm are considered non-contaminated. Altogether, in practice, a substrate with 300 ppm excess Pb was considered to be convenient for assessing biofilm effects on alleviating metal toxicity.

Before producing a contaminated substrate, the total amount of heavy metal needed to secure a 300 ppm concentration of bioavailable Pb was estimated. This is because Pb could interact with substrate components, being subsequently partly retained as a non-bioavailable fraction. Metal availability was assessed by adding increased concentrations of PbCl₂ to the substrate, thoroughly mixed, and left to stabilize for one month. Bioavailable Pb was determined after using the diethylenetriaminepentaacetic acid (DTPA) solution, as described by Page [47]. No retention of Pb by the substrate was noted, so the total amount of Pb added as PbCl₂ was recovered as bioavailable Pb. Then, the substrate was prepared with in excess of 300 ppm total Pb for its use in the contaminated treatments.

2.5.2. Biofilm Inoculation and Experimental Setup

Biofilm seed inoculation was carried out in treatments I and I + Pb. Again, biofilm and seeds were carefully mixed to ensure sufficient contact. In both biofilm-inoculated and non-inoculated assays, three plots per treatment were established, and 25 seeds per plot were sown. Each seed was grown in cells of 5 cm in diameter × 7.5 cm depth (approximately 0.15 L), which were filled with the substrate corresponding to each treatment. Each of the 25 seedlings in every plot was transplanted into 7 L pots. Then tomato plants were grown for 4 months. Greenhouse assays were carried out during the austral spring and summer period, i.e., from October to April. Average greenhouse temperature was 30 ± 5 °C. Substrate moisture was kept near field capacity by irrigating pots with tomato plants two/three times per week.

2.5.3. Plant Growth and Fruit Production Evaluation

To evaluate plant growth root and shoot biomass, plant height and leaf area (LA) were measured. Also, fruit production and fruit quality were assessed. Root and shoot biomass were calculated after drying at 70 °C to constant weight. Plant height and leaf area (LA) were quantified in each of the 25 plants of every treatment. Plant height was directly measured from the base of the stem to the upper part of the canopy. The leaf area of each plant was calculated from measurements made on 10 entire and fresh leaves, which were sampled at the central part of the canopy. The size of the selected leaves was medium, and a single leaf per branch was collected. Each leaf was photographed, and then its area was appraised by the IMAGE J software 1.8.0. [48]. Production of tomato fruit was calculated by counting tomato numbers and weighing each of them. Fruit quality was gauged in the fruit juice by the determination of total soluble solids (°Brix) by means of a handheld refractometer.

2.6. Lead Concentration on Tomato Fruit

To determine lead on tomato fruit, a standard acid digestion protocol followed by spectrometry was used. Sample preparation included air drying first and then oven drying. Next, samples were thoroughly washed to eliminate potential soil particles and impurities, and they were finally ground to a fine powder with a pestle and mortar.

Mineralization was carried out in a microwave digestion system (CEM model MDS-2000, American Laboratory Trading, East Lyme, CT, USA) using NO₃H and HCl, as described in the standard US-EPA SW 846-305 protocol [49]. The ground sample (0.5 g) was accurately weighed in a Teflon PFA vessel and treated with NO₃H and HCl reagents. Vessels were placed into the microwave oven for digestion. Thereafter, they were diluted to 50 mL in a volumetric flask and filtered through a 0.45 μm pore size filter. The Pb concentration in tomato fruit samples was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) in an ELEMT XR (Thermo-Finnigan, Somerset, NJ, USA).

2.7. Experimental Design and Statistical Analysis

A one-way analysis of variance (ANOVA) was used to determine differences of means between treatments. Soil germination, plant production, and fruit quality were analyzed according to a completely randomized factorial model with two factors (inoculation and substrate excess Pb). The Tukey test (p < 0.05) was used to identify statistically significant differences.

3. Results

3.1. Impact of Pb Added to Liquid Medium on Bacterial Growth

The efficiency of a bioinoculant lies in the capacity of the specific microorganism to maintain its viability in the substrate where the seeds will develop. This will ensure functionality in promoting plant growth even under adverse conditions. Therefore, the ability of Bacillus subtilis subsp. spizizenii to grow with increasing concentrations of Pb, including those considered potentially toxic, was investigated, highlighting its importance as a tool in sustainable agriculture against heavy metal contamination.

Figure 1 shows the bacteria growth curves expressed by the evolution of OD_610mm as a function of time. Typical latency (lag), exponential and stationary phases are recognized; also, the initial part of the death phase becomes apparent. The studied strain of B. subtilis was able to grow in Pb-containing medium at all concentrations tested. At the lowest Pb concentration tested (50 ppm), bacterial growth at the stationary phase was similar to that observed in the absence of the metal. However, for all the studied Pb concentrations, the latency phase was prolonged up to 30 h with respect to the control treatment with no Pb. Subsequently, in the logarithmic growth phase, the slopes of all curves were similar, except for the control. In the stationary phase, as Pb concentration increased the optical density (OD) decreased, reaching its lowest value of 1.84 corresponding to 300 ppm, which represents a 15% reduction compared to bacterial growth in the control without Pb. Furthermore, at 300 ppm Pb, the death phase was more pronounced than at the other concentrations studied.

Many of the metal detoxification mechanisms involve the synthesis of specific proteins as well as other types of compounds. Therefore, the prolongation of the latency phase could be due to the fact that the bacteria must first synthesize some of these molecules in order to adapt to the new environment with excess Pb.

3.2. Impact of Pb on Biofilm Production

Using a straightforward and easily available carbon source, such as 1% glycerol together with L-glutamic acid as a nitrogen source, the production of biofilm obtained at the Erlenmeyer air/liquid interface was 1.35 mg biofilm/mL of culture (Figure 2). The biofilm was sufficiently firm and adherent to be easily recovered using an elementary manual method such as a glass rod. The addition of 50 ppm Pb to the culture medium increased biofilm production with respect to the control, but this gain was not significant (p < 0.05). However, higher concentrations of Pb, that is, 100, 200, and 300 ppm Pb, significantly (p < 0.05) boosted biofilm production until figures near 1.5 mg biofilm/mL, which corresponded to about a 15% increase over the control.

3.3. Impact of Pb and Temperature on Biofilm Stability

To analyze the stability of the biofilm its production along time was evaluated, during a period sufficient for culture degradation. The culture was grown in a MSM added with 55 mM L-glutamic acid plus 1% glycerol as a carbon source. Figure 3 and Figure 4 show the biofilm obtained under four different Pb treatments (50, 100, 200, and 300 ppm) and the control treatment at 24 °C and 4 °C, respectively. At room temperature (24 °C) the biofilm was stable for 25 days, then gradually began to disintegrate until its total disintegration that took place at 45 days (Figure 3). At 4 °C, however, the biofilm was stable for 35 days. Then the degradative processes started and culminated in its total disintegration at 60 days (Figure 4).

From a nutritional point of view, for microbial growth, glycerol can replace traditional carbohydrates such as glucose, sucrose, and starch in some fermentation processes. In this work, pure glycerol was chosen as a substrate for bacterial growth because the use of an impure carbon source could cause toxicity problems. In the future, it is our intention to use this substrate with fewer purification processes in order to contribute to the utilization of this product that accumulates without final disposal.

Comparing the times required for biofilm disintegration at both 24 °C and 4 °C, a decrease of enzyme activity at low temperatures is clearly patent. The reduced enzymatic activity at 4 °C contributed to slowing down the degradation processes so that the biofilm remained stable for a longer period of time (Figure 4 and Figure 5). With the addition of different concentrations of Pb, both at 24 °C and at 4 °C, the biofilm yield over time was not significantly different compared to that of the control treatment without metal. This suggests that although the presence of Pb somehow stimulated a higher biofilm production, the presence of the metal did not affect its stability. Biofilm adhesiveness together with its ability to remain intact for a prolonged period of time are two important factors. Both would ensure an intimate contact between the biofilm and the seed, which could explain its high effectiveness as a growth promoter. This close contact would increase the chances of colonization by B. subtilis and facilitate the growth regulators released by the bacteria (type AIA) to benefit the initial stages of germination and seedling establishment.

3.4. Impact of Pb and Biofilm Inoculation on Seed Germination

Figure 5A,B show the relative germination percentage (RG%) and the relative root elongation (RRE%) of tomato seeds, respectively, for the control treatment and the four treatments with increasing Pb concentrations. Non-inoculated seeds exposed to the highest Pb concentrations (200 and 300 ppm) showed a decrease in germination percentage (RG%) of approximately 35% with respect to the control (Figure 5A). However, biofilm-inoculated seeds partly reversed this negative effect so that the decrease in RG% was limited to about 20% for seeds exposed both to 200 and 300 ppm Pb (Figure 5A).

Relative root elongation (RRE %) showed important variations as a function of Pb concentration. Seeds growing on a substrate with 50 ppm Pb showed a 21% higher RRE % compared to seeds growing on a metal-free substrate, and this for both inoculated and non-inoculated seeds (Figure 5B). At concentrations above 50 ppm, however, Pb produced a toxic effect. In particular, at 100 ppm Pb, root elongation in non-inoculated seeds decreased by 15%, while at 200 and 300 ppm, the reduction of this parameter reached 45%. The presence of a stimulatory effect on RRE at low Pb doses together with an inhibitory effect at high Pb doses indicates a hormesis phenomenon. Biofilm seed inoculation showed a beneficial effect on RRE at 100, 200, and 300 ppm Pb, mitigating the metal’s toxic effect. Thus, in the case of seeds exposed to 100 ppm Pb, the reduction in root elongation was only 10%, while at concentrations of 200 and 300 ppm Pb, this reduction was only 20% (Figure 5B).

Figure 6 shows the germination index (GI%) as a function of Pb concentration for non-inoculated and inoculated seeds. This is an index estimated from RG% and RRE% and associated with toxicity levels, therefore allowing evaluation of excess heavy metal impact on seeds. The effects of Pb concentrations observed for relative germination and relative root elongation are reflected in the germination index (GI). Higher germination index values are obtained for seeds growing at the solution with 50 ppm Pb. These values are greater than those obtained in seedlings growing in the absence of metal, which may be due to the effect of hormesis. When seeds were exposed to 200 and 300 ppm Pb and inoculated with biofilm, a GI of 64% was recorded. In contrast, non-inoculated seeds showed a GI of 35.7% (Figure 6).

Toxicity of soil heavy metals is an important factor in plant development because it can alter the highly sensitive stage of seed germination, impairing plant growth. As the GI % index considers the imbibition, emergence, and root elongation phases, the effect of Pb could be classified as non-toxic, moderate toxicity, or high toxicity. Accordingly, seeds germinating with 0 and 50 ppm Pb showed no toxicity. However, the GI% of seeds exposed to 200 and 300 ppm Pb corresponded to ‘moderate toxicity, while the GI% of non-inoculated corresponded to ‘acute toxicity’ (Table 1, Figure 6).

3.5. Tomato Plant Growth and Fruit Yield and Quality Under Greenhouse Conditions

3.5.1. Plant Root and Shoot Agronomic Parameters

Several agronomical parameters were measured at harvest time on the tomato plants of the different treatments growing under greenhouse conditions. Figure 7A,B show root and shoot biomass, respectively, obtained at the four treatments studied, namely inoculated and non-inoculated seeds growing in substrates with and without excess Pb levels. The toxicity effects at a concentration of 300 ppm Pb in the substrate on root and shoot biomass are evident. Root biomass decreased by 50% while shoot biomass decreased by 24% with respect to the control (Figure 7A,B, respectively). Root decay would restrict the volume of soil the plant can explore for water and nutrient withdrawal, which would negatively affect the overall development of the whole plant.

Biofilm seeds inoculation had a positive effect on both root and shoot biomass. In the substrate without Pb, seed inoculation increased the root and shoot biomass by 59% and 48%, respectively. for the aerial part. Similarly, in the substrate with Pb, seed inoculation rose by 135% and 43% for the shoot and root biomass, respectively (Figure 7A,B). Noteworthy, the treatment with excess Pb plus seed inoculation exhibited similar root and shoot biomass as the control treatment without inoculation. Therefore, the application of biofilm reverted the negative effects of Pb contamination on the substrate.

In the conditions of this work, B. subtilis produced a greater amount of biofilm with Pb additions (Figure 2). Therefore, it could be hypothesized that the adsorption of Pb⁺² ions by the functional groups of the exopolysaccharide (EPS) matrix together with specific electrostatic interactions could be one of the mechanisms used by the bacteria to prevent the metal from entering the plant. Moreover, taking into account that the bacterium was able to grow in a liquid medium with high levels of Pb (Figure 1), other different mechanisms would be involved in the uptake and bioaccumulation of the metal.

Figure 8 shows plant height and leaf area for the four treatments studied. In non-inoculated treatments, plant height was significantly (p < 0.05) affected by the presence of Pb, with a decrease of 15.7% compared to the control (Figure 8A). Seed inoculation with biofilm showed a positive effect on plant height in plants growing on treatments with and without metal. In the case of plants growing on substrates with excess Pb, the increase in height promoted by seed inoculation was 17%, while in the treatments without Pb, the increase was 22% (Figure 8A). However, excess in the substrate Pb has no effect on leaf area (Figure 8B). This notwithstanding, seed inoculation increased leaf area by 26% and 36% in the case of plants growing in the presence and absence of Pb, respectively (Figure 8B).

The results obtained for leaf area and plant height are consistent with those obtained for aboveground and root biomasses, showing significantly higher values in plants treated with biofilm (Figure 7A,B). Additionally, it is important to state that the leaves of plants growing in the Pb-contaminated substrate were less turgid than their counterparts, not impacted by Pb excess.

The results presented in this section provide evidence showing that inoculation of tomato seeds with biofilm obtained from B. subtilis subsp. spizizenii increases plant growth and production, even under stress conditions imposed by excess Pb in the substrate. Therefore, biofilm inoculation can be considered a tool to strengthen the environmental sustainability of tomato plants growing in contaminated substrates under greenhouse conditions.

3.5.2. Fruit Yield and Quality

In addition to plant growth, yield and quality of tomato fruit, are key aspects, given the importance of the edible part of this plant for human consumption. In this work, tomato production was quantified by the number of fruits and the fruit weight, while quality was assessed by a specific index, the Brix index. Figure 9 shows fruit number, fruit weight, and Brix index of the four treatments studied. Excess Pb in the substrate leads to a decrease in the fruit number, which was not significant in the non-inoculated treatment and significant in the inoculated treatment (Figure 9A). The effect of inoculation on fruit number was positive, resulting in an increase of 50% with respect to non-inoculated treatments for plants growing both on substrates with and without metal excess (Figure 9A).

Pb severely affected fruit size with a decrease of 26% in the mean fruit weight with respect to the control. Inoculation increased fruit weight by 42% and 39% in the cases of plants growing with excess and without excess Pb, respectively. The fruit weight of the inoculated treatment affected by excess Pb was similar to that of the control treatment, showing the recovery effect of seed inoculation (Figure 9B).

The beneficial effect of the PGPB bacteria was also reflected in the soluble solids content (Brix), a key parameter for assessing tomato quality, especially in terms of sweetness and flavor. Again, Pb excess significantly (p < 0.05) decreased Brix value with respect to the control. However, seed inoculation significantly increased the Brix of tomato fruits both in plants growing on Pb-contaminated and non-contaminated substrates. Inoculated plants without exposure to metal toxicity exhibited an optimum value of Brix = 4, which is considered a reference. This increase in Brix induced by inoculation not only highlights the positive influence of the bacteria on fruit quality but also reinforces its potential to improve the market acceptance of tomatoes.

3.6. Pb Concentration in Tomato Fruits

The Pb concentrations on the tomato fruit of the four treatments studied are shown in Figure 10. Mean fruit Pb values range from 0.37 to 0.41 mgkg⁻¹, being higher than the maximum values recommended by FAO/WHO [4] and the European Commission [41]. No significant differences between treatments have been found (p < 0.05). Therefore, neither substrate contamination nor seed inoculation posed additional risk for tomato consumption. Indeed, the tomatoes collected on Pb-contaminated treatments exhibit a little higher Pb content than those growing in non-contaminated substrates, but differences are meaningless in practical terms. Moreover, no significant effect of seed inoculation on Pb content of tomato fruit was detected in our study conditions.

These results suggest that the uptake of excess Pb in the substrate is accumulated and stored in roots and/or shoots, but not in the edible part of the plant, which will be discussed later. Again, fruits of plants growing on contaminated substrates did not accumulate more Pb than the control. Therefore, even if urban contamination is perceived as a risk for tomato and other vegetable crops, substrate Pb contamination likely did not pose any additional risks than those derived from background environmental pollution.

The fruit Pb concentrations were also similar in plants grown from biofilm-inoculated and non-inoculated seeds. These results suggest that the growth-promoting effects of Bacillus subtilis subsp. spizizenii biofilms are not related to processes linked to the retention of the metal in the substrate, which would prevent its entry into the plant. On the contrary, this effect could be associated with the biofertilization mechanisms utilized by this bacterium, such as biological nitrogen fixation, phosphorus solubilization and the release of growth regulators such as auxins and cytokinins. These mechanisms, acting together, would favor the integral development of the plant.

4. Discussion

4.1. Impacts of Increasing Pb Concentrations on B. subtilis Performance

A longer latency phase, as in our work, has been reported by Sharma et al. [50] working with Bacillus cereus grown in a liquid medium with 1000 ppm Pb, which was extended by 20 h. However, an increase in the latency phase length in the presence of metal was not observed in Enterobacter faecalis [51]. During this phase of the bacterial growth cycle, cells are not dormant, and the synthesis of RNA, enzymes, and other molecules occurs. For example, metallo-regulatory proteins allowing active defense systems against toxic metals [52] have been documented. Also, cysteine-rich proteins, i.e., metallothioneins, have been isolated from several Bacillus species, which are responsible for metal uptake and storage [53,54]. In addition, bacteria possess buffer systems that protect them from exposure to toxic metals. These are low molecular weight molecules (such as amino acids, glutathione, and organic acids) that bind the metal, buffering its toxic effect [55]. Specifically, in B. subtilis, bacillithiol (BSH) is one of the main components of the buffer system [55].

In this study, increasing Pb amount in the culture medium increased biofilm production. In contrast, in a previous work [26], biofilm synthesis by Bacillus subtilis subsp. spizizenii under high concentrations of Zn drastically decreased under high Zn concentrations. A biofilm matrix is made up of more than 80% exopolysaccharides (EPS), a polyanionic structure [56]. Therefore, it has been suggested that the increase in biofilm formation, when the bacteria are exposed to increasing Pb concentrations, could be linked to an increase in the synthesis of EPSs [56,57].

Moreover, it has been shown that the interaction between metal ions and polyanionic EPSs occurs with different degrees of selectivity and affinity, indicating high adsorptive properties towards heavy metal ions [57,58]. During the metal binding process in EPS, carboxyl and hydroxyl groups form coordination bonds, which provide stability to the metal–biopolymer complexes. In particular, uronic acids account for more than 50% of EPS, and the carboxyl group is the main functional group responsible for the metal-binding capacity through electrostatic-type interactions [59,60]. Metal-tolerant bacteria were found to produce exopolysaccharides, and these polymers would be responsible for their tolerance to metal excess [61,62].

The adsorption of heavy metals by tolerant bacteria has been evaluated as a strategy for the bioremediation of metal-contaminated soils [63,64]. For example, the inoculation of maize seeds grown in soil with high Pb content with B. subtilis reduced the metal accumulation in the soil and its concentration in leaves, stems, and roots [65]. Also, the Pseudomonas strain sp. DSP17 was able to build up more biofilm when growing in the presence of Cu, Pb, and Zn, which would alleviate the metal damage to the plant [66].

It has also been reported that many of the competitive advantages of B. subtilis in the colonization of tomato roots are due to its ability to develop a biofilm that facilitates water retention and protects the roots from pathogenic microorganisms [67]. Additionally, L-malic acid, one of the components produced in root exudates, has been suggested to be a trigger for biofilm formation [68,69]. It is likely that other molecules in the rhizosphere can also fulfil this function. Interconnections between microorganism colonization capacity, biofilm development, and biocontrol activity have been observed not only in tomato plants but also in other plant species such as cucumber [70], cotton [71], banana [72], and arabidopsis [68].

Similar to other uncharged small molecules, glycerol migrates through the cytoplasmic membrane by passive diffusion. Notwithstanding, also an integral membrane protein called GlpF facilitates glycerol transfer to the cell [73]. Once inside the bacteria, it easily becomes a carbon source. First, it is converted into acetyl-CoA. Finally, it can either follow the Krebs Cycle route or be subjected to successive modifications until it is transformed into PHB [74]. In Argentina and elsewhere, currently, large quantities of crude glycerol are available as a by-product of biodiesel production. Commonly, they must be disposed of as waste, because the existing market cannot absorb the increase in this by-product [75]. Different strategies for glycerol depletion have been evaluated. For example, crude glycerol has been used as a substrate for the production of metabolites of microbial origin, such as organic acids, polyols, and lipids by fermentation. Also, the addition of crude glycerol to the culture medium has been used by Bacillus pumilus for the synthesis of 1,3-propanediol [76] and Klebsiella oxytoca for the synthesis of 2,3-butanediol [77].

In our work, the biofilm stability strongly depended on temperature but was not affected by Pb concentration in the culture media. Enzymes such as proteases, glycosidases, and DNAases have the potential to degrade the polysaccharides (EPS) at the biofilm matrix [78]. Furthermore, these enzymes can induce cell lysis and interfere with the biofilm’s own sensing signals [79]. Therefore, as the culture ages, cell lysis becomes more pronounced, and this in turn is accompanied by increased release of enzymes. Moreover, biofilm-associated proteins are vital for the maintenance of EPS stability [80], and their degradation at the expense of protease enzymes leads to a disruption in their structural integrity and their mass dispersal. The advantages of using biofilm as a new form of bioinoculation compared to conventional liquid inoculants, based on the adhesiveness and stability of the former, have been reported previously [26,81].

The addition of Co to the culture medium of B. subtilis showed that the bacteria keep growing but did not develop a biofilm. However, with the addition of Cu, there was even no bacterial growth [81]. The deleterious effect of these transition metals for Bacillus has also been observed in other bacteria such as Streptococcus pneumoniae and Klebsiella pneumoniae B5055 [82,83]. Other metals such as Fe and Ca have shown positive effects on biofilm development by significantly influencing its stability and mechanical properties. For example, in Pseudomonas aeruginosa Ca⁺² ions increased biofilm stability due to cross-linking with alginate-type polysaccharides [84].

4.2. Impacts of Increasing Pb Concentrations on Seed Germination

The variations on relative root length (RRE%) can be associated with the hormesis phenomenon Pb-induced, as a dose–response relationship is obvious [85]. In biology, hormesis is defined as an adaptive response of the cells of organisms to external factors, such as the presence of essential metals [86]. On the other hand, seeds inoculated with biofilm showed benefits in terms of increasing RG%, RRE%, and GI%. This could be explained by the fact that B. subtilis produces growth regulators such as indole acetic acid (IAA) [25], which has demonstrated favorable effects on the development of both the main root and secondary roots and absorbing hairs [87,88]. In a previous work we reported that inoculation of Lactuca sativa seeds with the biofilm produced by this bacterium resulted in increased root development [25].

Negative effects of Pb on the germination of other plant species on germination parameters have been reported. This is the case for Catharantus roseus [89], Triticum aestivum [90], Phaseolus vulgaris [91], and Hordeum vulgare [92]. In the case of Triticum aestivum [90], the reduction in germination induced by excess Pb could be attributed to the inhibitory effect induced on the activity of enzymes, such as amylases, proteases, and phytases. Similarly, decreases in α and β amylases, acid invertases, and acid phosphatases responsible for carbohydrate metabolism have been found [93]. It has also been quoted that the presence of Pb did not impact the germination of Zea mays and Cleome amblyocarpa, although root elongation was affected [91,94]. On the other hand, it has been argued that under certain stress conditions, such as exposure to excess metal levels, high temperatures, and decreased pH, seeds can interrupt their dormancy periods and germinate more quickly [95]; this effect was observed in the case with Elsholtzia argyi exposed to 83 ppm Pb [96]. Considering metals other than Pb, it has also been claimed that they can induce or not a negative effect on seed germination and root elongation. For example, lettuce seeds exposed to Zn showed a negative effect on germination percentage and a very marked decrease in root elongation [97]. Similarly, tomato seeds exposed to 175 ppm Cu showed a 47% decrease in germination percentage [98]. These results would suggest that the germination and plant growth response to metal stress would be specific to each plant species [99].

4.3. Impacts of Pb and Seed Inoculation on Plant Growth and Fruit Quality

The toxic effects of Pb on root and shoot biomass are consistent with results reported previously by Akinci et al. [100], showing a 35% and 34% decrease in tomato root and shoot biomass, respectively, when seeds were exposed to 300 ppm excess Pb. Moreover, Festuca arundinacea plants exposed to increasing concentrations of up to 1000 ppm Pb showed biomass reduction and also an increase in the plant’s antioxidant activity [101]. Also, the detrimental effect of Pb in relation to biomass decline has been observed in Elsholtzia and Zea mays. It has been pointed out that the most notable morphological alteration caused by heavy metal stress is the reduction in plant growth [98]. The efficiency of B. subtilis subsp. spizizenii biofilm as a new method of seed inoculation has already been proven in an experiment with tomato plants growing on substrate without Pb contamination. Biomass increases of over 100% for the root and 80% for the shoot were obtained [26].

The main route of Pb plant uptake is through roots, as a divalent cation. Therefore, it is expected that this pathway could affect plant nutrition due to a competition effect by inhibiting or delaying the uptake of other divalent cations, such as K⁺ [102]. In particular, for tomato, a decrease in the incorporation of essential elements such as Mn and Fe in presence of excess Pb has been observed [103]. This competitive effect has also been documented in other plant species. For example, exposing cauliflower (Brassica oleracea L.) to 200 ppm Pb led to a reduction of P, S, Fe, Mn, and Cu uptake [104]. Also, radish (Raphanus sativus subsp. sativus L.) submitted to 100 ppm Pb reduced Fe levels [105]. Moreover, an inverse correlation between K and Pb concentrations has been reported, which was attributed to the fact that both cations have almost equal radii. Thus, it has been suggested that Pb could affect K⁺ATPase channels and -SH groups of biological membranes, which in turn could lead to outflow of K⁺ in root cells [106].

Toxicity of Pb can also damage cell membranes, causing impacts such as stomata closure, which may lead to CO₂ deficiency and alterations in water uptake. Alterations at the membrane level also can lead to increased membrane permeability and cell lysis. Moreover, Pb uptake by plants induced a reduction in transpiration rates, thus stopping the flow of water and nutrients from soil to plant, resulting in growth suppression [103]. Specifically, in sunflower (Helianthus annuus L.), the reduction in transpiration rate boosted a water deficit, which induced proline synthesis in an attempt to counteract water stress [107]. The decrease in water absorption from the soil has been explained by the fact that water entry occurs particularly along certain areas of the root, namely the root apex, which in turn are the areas of greatest absorption of Pb because in these areas the cell walls are thinner, and the acidity of the medium enhances the solubility of the metal [108].

Several mechanisms have been proposed to explain the effect of Pb toxicity. The most robust include overproduction of reactive oxygen (ROS), modifications in root architecture, and production of exudates. ROS causes oxidative damage to cells [102,109]. Root architecture alterations include increased branching and greater curvature in root areas in contact with metals [110]. The production of exudates is considered the first line of defense of plants against heavy metals, as they reduce the entry of metals into cells by complexing with them. Other toxicity mechanisms include increased efflux of metals from cells to the outside as well as their biosorption into cell walls.

Soil microorganisms also control the heavy metal dynamics either directly or indirectly. For example, a high tolerance of Bacillus to the presence of metals in mine soils has been reported by several authors [111,112]. The ability of microorganisms to promote plant growth in soils with high levels of metals could be related to strategies promoting changes in the chemical speciation of the element of interest [113]. Some microorganisms have the potential to reduce metal ions to less toxic forms by changing their oxidation state. Also, anaerobic microorganisms can use these metal ions as terminal electron acceptors [50]. Metal bioaccumulation depends on several factors, such as the internal structure of the microorganism itself, its biochemical characteristics, its genetic capacity, and changes in the charges present on its cell surface, as well as depending on environmental conditions [114].

Responses to stress induced by excess heavy metals show alterations with different levels of complexity affecting plant height and/or leaf area. For example, a decrease in the cell wall plasticity, which causes plants to lose turgor pressure in occlusive cells and their stomata to close, has been observed [115]. Also, the water disturbances that occur in the plant due to the presence of Pb cause leaves with lower turgor [102].

This is associated with a decrease in the concentration of sugars, amino acids, and other molecules involved in the control of cell turgor. To counteract these effects, plants synthesize higher concentrations of osmolytes, especially proline [116]. It was also shown that in tomato plants exposed to Zn, leaf area and leaf number were significantly reduced, which may be a result of inhibition of cell division, elongation, or both [117].

Heavy metal stress promotes the development of symbiotic relationships between plants and various organisms. These interactions may generate beneficial effects on plants through various mechanisms such as biofilm formation, production of phytohormones, exopolysaccharides, and siderophores. These associations contribute to increased metal tolerance and biomass production [10]. The beneficial effects encountered after inoculation with biofilm of Bacillus subtilis subsp. spizizenii on tomato growth parameters could be attributed to the ability of the bacterium to act as a biofertilizer and phytostimulant. This is partly due to the release of indoleacetic acid (IAA) and cytokinins [25], which influence cell morphogenesis and proliferation. These compounds promote further development of the main root, lateral roots, and absorbing hairs, allowing the plant to expand its exploration area in the soil to improve its nutrition. Another mechanism associated with the potential use of biofilm as a biofertilizer relies on phosphorus solubilization [25]. Additionally, previous studies have shown that the bacterium can persist inside the roots over time, suggesting that its beneficial effect could be maintained throughout the crop cycle [26]. Other mechanisms promoting the use of Bacillus spp. as a biofertilizer include its ability to fix nitrogen [118] and produce siderophores [119].

In a previous study, the growth-promoting effect of biofilm produced by B. subtilis subsp. spizizenii on the yield of tomato plants growing in a substrate with excess Zn was measured. The results of this study showed the beneficial impact of inoculation both on the number and weight of tomato fruits and their soluble solids content [97]. The optimum value of 4 for Brix was established according to Santiago et al. [120], and it is considered as the reference standard for fresh tomato consumption. In other species, such as, for example, coriander, the presence of high levels of heavy metals in soils also has been reported as a source of poor fruit development [121].

4.4. Lead in the Tomato Fruit

As shown in our work, in general, plant concentrations of Pb are very low, even when grown on sites highly contaminated with this metal [3,6]. Plant species like tomato, whose edible part is the fruit, show the lowest risk of Pb contamination, while accumulation of Pb in shoots and roots of plants growing on contaminated soils or substrates has been widely reported [122,123,124]. Increased metal accumulation in plant roots appears to be a strategy to keep metal concentrations in aerial organs low. In the case of S. lycopersicum, this is especially beneficial since the fruits represent the edible part of the plant [122]. Moreover, plants have several strategies to counteract the negative effects of heavy metals. For example, if the metal comes into the root, plants can activate tolerance mechanisms such as compartmentalization of the metal in different intracellular niches or biosynthesis and accumulation of specific compounds able to start metal complexation [124]. This prevents metal transport from the root to other parts of the plant. The selected strategy will largely depend on plant genetics and growing conditions [125]. Our results showed that tomato fruit Pb concentrations in the Río Grande variety were above the established limits for consumption [4,41]. Similar results have been obtained before, and overall, this suggests a minor risk of Pb to consumers of tomato fruit [126,127]. Similar to our work, a few studies found higher Pb levels in tomato fruits than the maximum recommended values [125,128,129]. However, as previously stated, on the one hand, fruits of plants grown on Pb-contaminated soils or substrates do not show higher metal contents than their counterparts grown on control non-contaminated plots, and on the other hand, no risk to consumers in terms of Pb accumulation in tomato fruits has been found until now [125,126,127]. This notwithstanding, management practices reducing Pb accumulation in soil and uptake by tomato plants in urban areas are recommended.

5. Conclusions

Tomato seed inoculation with biofilm produced by B. subtilis subsp. spizizenii had a plant-promoting effect on tomato. Germination, plant biomass, and fruit quality were enhanced even under stress conditions imposed by Pb contamination in the substrate. Therefore, seed biofilm inoculation has been shown to be a tool to achieve the environmental sustainability of tomato plants grown in greenhouses.

Overall, our results suggest that the beneficial impacts produced by seed inoculation with biofilm of B. subtilis subsp. spizizenii on tomato plants growing in substrates with extra Pb were not linked to mechanisms associated with the external retention of the metal. Rather, the effective protection by biofilm seed inoculation of tomato plants grown in Pb-contaminated substrates could be roughly related to PGPBs, expected effects, for example, biological N fixation, P solubilization, etc.

Bacillus subtilis subsp. spizizenii showed high resistance to the presence of Pb, tolerating concentrations of 300 ppm of this metal. Inoculation of S. lycopersicum var. Rio Grande seeds with the biofilm produced by B. subtilis protected the seeds from the toxic effects of Pb during germination, decreasing the toxicity of the metal from high to moderate. At the end of the growing cycle of tomato plants grown in a substrate with excess Pb, the plants whose seeds had been inoculated with the biofilm showed a higher development in growth parameters, as well as in the number and soluble solids content of the fruits.

B. subtilis biofilm can be produced cost-effectively using biodiesel byproduct glycerol. Commonly, glycerol must be disposed of as waste because the increase in this product cannot be absorbed by the existing market. Therefore, the approach used in this work could be a worthwhile biotechnological solution for applying in soil remediation and for reducing the dependence on agro-chemicals, particularly pesticides. However, this approach still has not been implemented in sustainable horticulture in urban periphery environments, and therefore field experimentation is recommended. Because of the entophytic nature of B. subtilis and also due to the presence of spores in the biofilm, long-term persistence has been taken for granted, but this aspect as well as the long-term effect on soil microbiota needs further research.