**4. Discussion**

The long-term sustainability of food chains and the managemen<sup>t</sup> of high levels of food loss and waste are among the challenges the global agri-food system have been facing in recent decades [24]. Bread, whose predicted production volume in 2021 was 209,874.8 million kilograms [25], represents a large portion of the global food waste, with economic and environmental repercussions [26]. The valorization of bakery waste as a food ingredient has been largely investigated recently, and different innovative biotechnological protocols have been proposed aiming at obtaining glucose syrup [27,28] or beer [26,29]. Recently, bioprocessing, e.g., enzymatic treatments and microbial fermentation, have been used to convert bread waste into valuable food ingredients, aiming at the improvement of the technological and sensory characteristics of the biomass, but also to the in situ enrichment of functional compounds such as dextran (with a positive impact on food texture) [30], antimicrobial compounds [31], and GABA [32].

Bioprocessed wasted bread, thanks to its suitability to be converted into a substrate for the growth of several microorganisms, was successfully used for the production of a medium for food industry starter cultivation [8]. Nevertheless, a major part of the wasted bread is no longer edible, not eligible for human consumption, and therefore disposed of as waste, thus representing an environmental issue due to the very high organic load. Only a small part of the wasted bread is employed for ethanol production or re-used as feed [7,26].

In this work, the potential of wasted bread to be used as a soil amendment was investigated. In addition to untreated wasted bread, bread biomass pretreated with enzymes and fermented with selected lactic acid bacteria was considered.

Overall, valorisation of food waste by conversion into products such as biofertilizers and biochar that can be added to the soil for increased nutrient inputs and fertility is gaining attention by the scientific community and industry [33]. Food waste fertilizers can be a relatively cheap source of nutrients compared to commercial inorganic fertilizer sources due to their large availability and the possibility of mass-scale and low-cost production. In addition to the nutrient role of the organic biomasses in soil, food waste can sometimes act as soil amendments, since they are able to affect the PGPM growth and survival, reduce pathogens, release nutrients, reduce leaching, increase water retention, and improve soil structure [34,35]. It was already observed that, as soil amendments, food waste-derived biomasses can increase plant yield and soil productivity [36].

The role of PGPM in soil fertility appears to be crucial; nevertheless, past research only focused on a few groups of common symbiotic rhizosphere microorganisms, such as rhizobia, *Bacillus*, *Pseudomonas*, and mycorrhizal fungi [37]. The functional role of other groups of potential PGPM, including LAB, has not been largely investigated [12], although such microorganisms could represent a genetic and metabolic resource for the development of biochemical solutions to pressing agricultural issues [12]. LAB are ubiquitous members of many plants, soil, and compost microbiomes, but little is known about the functional interactions between the LAB and their hosts.

LAB were shown to solubilize phosphate [38,39], likely through the production of organic acids, and it was also hypothesized that they can fix atmospheric nitrogen [39] or produce siderophores [38]. LAB could act as biocontrol agents; through the production of antimicrobial compounds, reactive oxygen species, and bacteriocins; by excluding pathogens by pre-emptively colonizing plant tissues vulnerable to infection and by altering the plant immune response [12]. Among LAB features, of interest not only from an agronomic but also from environmental purposes, the ability of *Lactiplantibacillus plantarum* to absorb Ni2+ and Cr2+ (from industrial wastewater) on the surface and inside their cells was proposed by Ameen et al. [40]. The superficial adsorption is possibly due to the electrostatic interaction of metals with the functional groups of the bacterial cell wall [41].

In this framework, the inoculum of wasted biomasses with properly selected LAB could guarantee the dominance of the LAB compared to other microbial groups, thanks to their capability to rapidly acidify the substrate and to produce antimicrobial compounds. In particular, the bioprocessed wasted bread harboured a very high population of the starter *L. plantarum* H64. The strain, previously selected for the ability to biosynthesize GABA in a matrix composed of wasted bread and wheat bran, allowed the repurpose of two of the main by-products of the cereal industry, promoting their application as a bread ingredient [32]. As expected, because of the starter carbohydrate metabolism, the bioprocessing enabled the production of organic acids which are in line with those previously reported in fermented surplus bread matrices [31,32]. On the contrary, proteolysis was not as pronounced if compared to common flour, which is explained by the fact that the proteases of the original flour, composing the bread dough, are degraded during the baking process. Indeed, unlike dairy LAB, most sourdough lactobacilli do not possess a cell-envelope-associated proteinase and depend on cereal-associated proteinase [42]. Hence, in surplus bread matrices, to ease the release of peptides available during LAB fermentation for their catabolism to amino acids or small bioactive sequences, the use of proteases should be considered, as previously reported [31,32]. Although a decrease in the total free amino acid content was observed in bioprocessed wasted bread, GABA content was 28% higher in bWB compared to WB. Additionally, one of the main advantages of the use of fermentation is the ability to prevent the proliferation of other microorganisms, either bacteria or molds, potentially spoiling bread. Indeed, a significant reduction of

yeasts, mold, and *Enterobacteriaceae*, was observed after bioprocessing. This is an aspect particularly appealing in terms of the industrial application of bioprocessed biomass since it can guarantee a longer shelf-life of the amendment.

To better understand the principal changes occurring during cultivation, the biomasses were characterized for their main physicochemical properties. The relatively high EC values observed for WB and bWB are probably related to the presence of sodium chloride, commonly added to the bread formulation at 1–2% (*w*/*w*), however, bioprocessing slightly but significantly reduced the EC value of WB, most likely a consequence of sodium lactate formation in presence of a high concentration of lactic acid produced by LAB metabolism. Wasted bread bioprocessing also led to a slight but significant increase and decrease of OC and TN content, respectively. As a result, fermentation determined a positive balance between the C fixed in microbial biomass and the C lost in heterolactic fermentation as CO2, whereas a major N loss as NH3 through LAB catabolic pathways involving free amino acids could be responsible for the lower TN content in bWB compared to WB [43].

When the biomasses were used as amendments, the soil pHH2O decreased because of the organic acids brought especially by bWB. The higher EC value of the biomasses reflected on that of treated soils, in fact, biomass mineralization could potentially release osmotically active compounds that could have contributed to the EC increase.

The use of biomasses raised the soil OC and TN content compared to the unamended soils. Among cultivated soils, CTP showed the lowest TN content at the end of the trial because of the uptake of nitrogen from the crop against no input. Since the physicochemical parameters (pH, EC, OC, and TN) showed the same trend in cultivated and uncultivated pots, it is safe to assume the biomasses, rather than the plants, were responsible for such changes. On the contrary, the availability of P was influenced by the crop since the absence of plants in amended pots resulted in a reduction of the Pava content compared to all other treatments. The possible explanation for such behaviour is that the application of biomasses enhanced the microbial activity resulting in the immobilization of phosphate as microbial biomass and phytate, the dominant organic P form in soils, that accumulates due to the deficiency of hydrolytic enzymes and precipitates with metal ions [44]. In contrast, the presence of the plants produced a rhizosphere effect which provided phosphatases, responsible for the solubilization of organic P, and suitable organic acids that compete with phosphates for the sorption sites [10]. Indeed, among organic acids the most efficient in solubilizing soil P are the di- and tricarboxylic ones, mainly oxalic and citric acid, while *L. plantarum* H64 employed in the present study mainly produced monocarboxylic acids, such as lactic, acetic, and γ-aminobutyric acids. The lower pH and the higher organic acid content also led to a major availability of Mn, Fe, and Cu in amended soils. These elements, through the ligand exchange, were solubilized from their precipitated oxides, as a consequence of the biomass's addition as well as the soil microbial community and rhizosphere activities [9].

To evaluate whether soil improvements transmuted to beneficial changes in the plants, escarole growth and composition were monitored. Biometric parameters of escarole plants indicated that WB and bWB promoted plant growth. Even though during the first 25 DAT no significant differences were observed among treatments, probably because of the rooting and establishment of the plants, the following days revealed a trend. Soil amended with bWB was the first to prompt a better chlorophyll content compared to the other treatments because of its higher TN content and the larger supply of GABA that is correlated to several beneficial effects for plants. Indeed, GABA was found to (i) defend roots against pathogens, (ii) serve as an N reservoir, (iii) cryoprotect the tissues, and (iv) synthesize plant hormones [45]. In contrast, the control plants showed the lowest significant SPAD values from 27 DAT until the end of the trial presumably due to nutrient limitation.

The elemental composition of escarole leaves did not reflect the soil availability of the studied elements. In fact, CTP plant leaves showed a P content more than double that of WB and bWB possibly due to the immobilization of P in organic matter. Regardless, this result did not negatively influence the yield of the crop but rather the nutritional

value of escarole. To avoid this inconvenience, transplanting should occur later than soil amendment to allow for better mineralization of the organic P. Regarding Mn, Fe, and Cu, their leaf content did not change among treatments although their soil content was higher in amended pots. It is hypothesized that their soil availability was already satisfied in the CTP pots since they are micronutrients, and/or those elements have been accumulated preferentially in the roots.
