Enriching NPK Mineral Fertilizer with Plant-Stimulating Peptides Increases Soilless Tomato Production, Grower Profit, and Environmental Sustainability

The need to increase agricultural production to feed a steadily growing population may clash with the more environmentally friendly but less efficient production methods required. Therefore, it is important to try to reduce the use of chemical inputs without compromising production. In this scenario, natural biostimulants have become one of the most sought-after and researched technologies. In the present study, the results of a greenhouse experiment on hydroponic tomatoes (Solanum lycopersicum L.) are presented, which involved comparing the use of ordinary NPK fertilizer (Cerbero®) with the use of NPK fertilizers enriched with 0.5% protein hydrolysate of plant origin (Cerbero Green®) at both standard (100%) and reduced (70%) fertilization rates. The results highlight how the use of Cerbero Green® fertilizers improves the production performance of tomatoes. More specifically, they show that the use of Cerbero Green® leads to higher marketable yields, especially under reducing fertilizer use, ensuring a positive net change in profit for the grower. In addition, carbon footprint analysis has revealed that the use of Cerbero Green® reduces the environmental impact of hydroponic tomato growing practices by up to 8%. The observed higher yield of hydroponically grown tomatoes even with reduced fertilization rates underlines once again the key role of natural biostimulants in increasing both the economic and environmental sustainability of horticultural production.


Introduction
Technological progress has driven the agricultural world towards a strong intensification that has, over time, reduced the number of malnourished people, generated employment, and income for farmers [1].However, the depletion of genetic potential and cultivable areas, coupled with population growth and sudden and unpredictable climate change, has imposed continuous new challenges on the agricultural sector [2][3][4].One of these is the reduction of energy consumption by improving the efficiency of use of invested resources.Indeed, the reckless application of chemical inputs, such as pesticides and chemical fertilizers, is no longer sustainable due to their serious impact on the environment and human health [5].Furthermore, it should be noted that some fertilizers used are produced from rock deposits that represent a non-renewable resource [6].Therefore, the productive agricultural world must 'move' towards sustainable development that, by definition, integrates the three dimensions of natural-human systems, namely economic, social, and environmental [5,7,8].To date, the necessary increase in agricultural production Plants 2024, 13,2004 2 of 16 cannot be dissociated from the assessment of nutrient use efficiency (NUE).The excessive use of chemical fertilizers has a cost, both in environmental (ecological footprint, water eutrophication) and economic terms associated with their production, transport, and application [9,10].For these reasons, it is necessary to maximize NUE to ensure environmental sustainability and economic viability.Specifically, the estimation of NUE is based on two key points as follows: (i) nutrient uptake efficiency, which takes into account the acquisition, inflow, and transport of nutrients into the roots, and (ii) nutrient utilization efficiency, again strongly influenced by the type of crop [11,12].However, NUE is influenced by a complex and multifaceted set of factors that, in addition to the aforementioned crop characteristics, also takes into account the chemical and physical properties of the soil, climatic parameters, and agronomic management aspects.Therefore, the much-discussed improvement in NUE in plants can only be achieved through careful manipulation of these five key factors.Over the past decades, several technological innovations that have been identified and studied can improve the sustainability of the agricultural world, many of which have been based on increasing NUE [13,14].The use of biostimulants, which include both natural substances and compounds (e.g., algae extracts, humic acids and protein hydrolysates) as well as beneficial microorganisms (e.g., rhizobacteria and mycorrhizal fungi) are among the most interesting strategies as they ensure that crop yields and quality are improved in a sustainable manner (e.g., by improving NUE) [15].At the European level, the economic value of the biostimulant industry is estimated at between 200 and 400 million euros (with an average annual growth of around 10%) [16].Among the various biostimulants, vegetal-derived protein hydrolysates (V-PH) have carved out a prestigious place for themselves in the world of horticulture.In line with the increasingly discussed concepts of circular economy, the production process used to produce these biostimulants (enzymatic hydrolysis) would fit well with agricultural organic waste, transforming it from a problem to be disposed of into a real economic benefit for farmers [16].Vegetal-derived PHs, besides containing limited amounts of macro-and micro-nutrients, are a rich source of soluble peptides and free amino acids that are mainly responsible for the biostimulating action of these products [17].Root or foliar applications of plant-derived PHs can trigger the activation of several physiological and molecular mechanisms in different crops, stimulating vegetative growth and resource-use efficiency and consequently improving yield and functional quality [18,19].For instance, it has been demonstrated that application of the V-PH 'Trainer ® ' on greenhouse crops is able to stimulate nutrient uptake and assimilation, with a significant increase in crop productivity [19,20]; this has been linked to the presence of amino acids and small peptides in the biostimulant product, which act as signaling compounds eliciting auxin-and/or gibberellin-like activities on both leaves and roots and thus causing a "nutrient acquisition response" that increases nutrients acquisition and assimilation as well as an increase in the photochemical efficiency and activity of photosystem II [20].
Furthermore, the stimulation of specific protective processes related to osmotic regulation and antioxidant activity provides PH-treated plants with increased 'protection' against a wide range of abiotic stresses [21].Foliar applications of vegetal-derived protein hydrolysate have proven to be able to reduce environmental impact of greenhouse spinach production, as CO 2 equivalent emissions per unit of spinach yield, especially under reduced nitrogen fertilizer rates [22].However, there is a lack of information about the effect of vegetal-derived protein hydrolysates on the environmental impact of other important vegetable crops such as greenhouse tomato.Moreover, the biostimulant applications also need to provide appropriate economic profit and competitive advantage for farmers.A previous study on greenhouse tomato demonstrated that foliar applications of vegetal-derived biostimulants enhanced fruit yield, leading to an increase in gross returns that ultimately improved the net returns as compared with the untreated plants [23].
Starting from the above considerations, a greenhouse trial was carried out to evaluate the impact of enriching NPK mineral fertilizers with a V-PH containing plant stimulating peptides on soilless production of greenhouse tomato, environmental indicators, and economic profitability.Environmental indicators were determined only for the first trial using the Life Cycle Assessment, following a cradle-to-gate perspective (plant cultivation phase up to harvest) considering both the direct emissions of the different phases of the process and the indirect emissions associated with the production of raw materials as inputs in the production chain.Moreover, economic profitability, associated with the replacement of NPK fertilizers with NPK fertilizers enriched with plant stimulating peptides, was assessed by partial budget analysis which focuses only on the changes in income and expenses that result from implementing a specific alternative.

Agronomic Results
The differentiated fertilization management proposed led to significant variations in all yield parameters reported in Table 1.Specifically, for both tested fertilization levels (100% and 70%), the use of NPK fertilizers enriched with 0.5% V-PH (Cerbero Green ® ) resulted in an average increase of 7.3% in fruit yield.A similar trend was partially observed for the number of fruits as well.Indeed, exclusively for plants fertigated at the 100% level, the use of Cerbero Green ® compared to Cerbero ® resulted in a significant increase (+5%) in this parameter (Table 1).Regarding the average weight of fruits, the use of Cerbero Green ® at 70% recorded significantly higher values compared to those obtained with Cerbero Green ® at 100%.

LCA Results
The tomato production results varied across different treatments.The highest yield was observed in plants treated with NPK fertilizers enriched with plant stimulating peptides and decreased mineral fertilization (Cerbero Green® 70%).This was followed by plants treated with Cerbero Green® 100%, and plants fertilized with decreased mineral fertilization without biostimulant application (Cerbero® 70%).The lowest fruit yield was obtained from Cerbero® 100%.Considering the functional unit as 1 ton of harvested crop, any increase or decrease in fruit yield had an inverse impact on all environmental indicators.
The findings presented in Table 2 demonstrated that the use of NPK fertilizers containing plant stimulating peptides led to reduced environmental impacts per ton of marketable tomatoes, regardless of whether standard or decreased fertilization was applied.For example, the use of Cerbero Green® resulted in a higher reduction of CO 2 emissions (−8%) in the plants subjected to decreased fertilization rate compared to those grown under standard fertilization (−5%).Applying protein hydrolysate with standard fertilization treatments resulted in a 4% to 11% decrease in all impact categories, while with decreased fertilization, the reduction ranged from 5% to 9%.Analyzing the process contributions to the total carbon footprint (Figure 1), it was evident that greenhouse heating due to natural gas consumption was the primary contributor, accounting for 80-82% of the total impact.Other significant environmental burdens were attributed to the production processes of peat-based substrate and mineral fertilizers, as well as on-farm emissions from consumption of these fertilizers, which had substantial impacts on various environmental indicators.
Plants 2024, 13, x FOR PEER REVIEW 5 of 5 treatments resulted in a 4% to 11% decrease in all impact categories, while with decreased fertilization, the reduction ranged from 5% to 9%.Analyzing the process contributions to the total carbon footprint (Figure 1), it was evident that greenhouse heating due to natural gas consumption was the primary contributor, accounting for 80-82% of the total impact.Other significant environmental burdens were attributed to the production processes of peat-based substrate and mineral fertilizers, as well as on-farm emissions from consumption of these fertilizers, which had substantial impacts on various environmental indicators.

Economic Results
The additional marketable yield resulting from the replacement of Cerbero ® with Cerbero Green ® was 2.7 and 5.3 t/ha for 100 and 70% level, respectively (Table 1).Therefore, the increases in tomato yield with Cerbero Green ® 100% and Cerbero Green ® 70% led to an additional gross yield of $2970 and $5830 per hectare, respectively (Table 3).Taking into account the total variable costs associated with the use of Cerbero Green ® 100% and Cerbero Green ® 70% (Table 3), the net change in profit for plants treated with Cerbero Green ® 100% and Cerbero Green ® 70% compared to those treated with Cerbero ® 100% and Cerbero ® 70% was $2365 and $4750 per hectare.The tomato selling price of 1.10 $/kg was used in the calculation of gross margin.The added variable costs include the additional cost resulting from the extra cost of replacing Cerbero ® with Cerbero Green ® fertilizers and the additional cost for harvesting the supplemental yield gained with Cerbero Green ® application instead of regular Cerbero ® .

Discussion
While the literature emphasizes the importance of genotype, environmental conditions, and their interactions on the effects of biostimulants [23][24][25][26], our experiment on tomatoes grown in soilless culture revealed a consistent positive response to biostimulant usage.The application of the Cerbero Green ® fertilizers, regardless of the fertilization level (100% and/or 70%), significantly increased the fresh yield of tomatoes, compared to the Cerbero ® fertilizers alone.It is noteworthy that the increased production of plants fertigated with Cerbero Green ® was primarily attributed to a higher number of fruits per plant, partially confirming what was observed by Rouphael et al. [27].These results could be attributed to the presence of specific bioactive peptides in Cerbero Green ® , which have a hormone-like activity in promoting rooting, plant growth, flowering and fruit set.Moreover, vegetalderived protein hydrolysate (V-PH), besides being characterized by the presence of amino acids and bioactive peptides, may contain traces of other useful compounds such as mineral elements, carbohydrates, phenols, and phytohormones [21].Although the mode of action of these plant biostimulants is still not completely known today, an increasing amount of data available in the literature highlights the positive effect of V-PH application on the regulation of critical phenological phases such as flowering and fruiting [21,27,28].
The increased number of fruits per plant could be directly related to a better response during the pre-fruiting phase, especially in protected environments where high daytime temperatures can negatively impact this process [27,28].Additionally, previous studies have shown that V-PH application has a direct impact on root system architecture [29,30].The improvement in the main parameters related to root architecture (total root area and root length) is directly linked to increased nutrient utilization efficiency and consequently to crop productivity.Specifically, the presence of auxin precursors, root-promoting peptides, and amino acids such as L-glutamate and tryptophan stimulate root growth and the development of absorbing root hairs through specific mechanisms not yet fully understood [27,31].This improvement in root architecture forms the basis of the efficacy of non-microbial plant biostimulants on nutrient absorption efficiency [32].
The notion that higher fertilization leads to greater production contrasts with current agronomic trends promoting more sustainable practices [33,34].Excessive fertilizer not only has a negative impact on the environment but also entails high management costs [35,36].In our study, the use of a lower fertilization level (70%) did not result in reduced yields compared to using Cerbero ® at 100%, highlighting the possibility of reducing fertilizer inputs.Furthermore, plants fertigated with Cerbero Green ® at 70% showed the highest yields, confirming the effectiveness of biostimulants in reducing mineral input [37][38][39].
Several Life Cycle Assessment (LCA) studies have investigated greenhouse tomato production.Studies indicate the importance of heating systems and energy consumption in global warming potential [40].Therefore, to facilitate comparisons, the studies have been categorized into heated and unheated greenhouse tomato productions.For unheated greenhouse tomato production, Sanjuan-Delmás et al.
[41] found that cherry tomato production in an innovative unheated rooftop greenhouse resulted in approximately 580 kg CO 2 equivalent per 1 ton of tomato.Similarly, Romero-Gámez et al. [42] estimated that tomato production in an unheated greenhouse could lead to approximately 617 kg of CO 2 per ton of tomato.In our study, excluding heating demand and corresponding impacts, the result ranged from 318 to 367 kg CO 2 per ton of tomato.
On the other hand, when considering heated greenhouse tomatoes, the outcomes were primarily influenced by the type of fuel used and the amount of energy consumption.Maham et al. [43] examined the environmental performance of greenhouse tomatoes heated by an electric heater under different levels of organic fertilizers and water stress.They estimated an average of 269 kg CO 2 per ton of tomato.Another study by Bosona and Gebresenbet [44] reported that tomato production in a heated greenhouse using woodchips could entail approximately 547 kg CO 2 equivalent per ton of crop.However, in our current study, the results were higher, ranging from 1754 to 2029 kg CO 2 per ton of tomato.This difference can be justified by the application of a natural gas boiler as the heating system in our study.
Overall, four treatments were implemented, each replicated four times, with five plants per replication.The complete fertilization plan is detailed in Table 5.Both NPK fertilizers (Cerbero ® and Cerbero Green ® ) were manufactured by Hello Nature Inc. (Anderson, IN, USA).Fertigation was performed using a drip irrigation system having one emitter per plant of 2 L/h.Fertigation was managed to assure that at least 30% of the drainage from the bags to avoid salt build up into the substrate.Pests and diseases were controlled by commercial pesticides at the labelled rates.

Inventory Data Collection
The experiment lasted 169 days (from 11 April to 27 September).At each harvest, the fruits from each treatment were counted, weighed, and separated into two groups, namely non-marketable fruits (green and/or deformed) and marketable fruits (free of visible defects and mature), to determine the marketable fruit yield, fruit number, and fruit mean weight.Moreover, all inputs, like peat-based substrate, mineral fertilizers, protein hydrolysate, irrigation water, pesticides, plastic, lubricant, heating, used in the cropping cycle were recorded and used for calculation of environmental indicators and carbon foot printing.
The necessary data for modeling the greenhouse tomato product system, particularly foreground data, were obtained from an experimental farm affiliated with Tuscia University.These foreground data encompassed critical information such as fertilizer quantities, seedling numbers, pesticide usage, and water consumption pertinent to tomato cultivation.
In contrast, background data concerning the production of input materials such as energy, seeds, and mineral fertilizers were sourced from the Ecoinvent database.This dataset contributes to offering a comprehensive understanding of the environmental impacts associated with the entire life cycle of greenhouse tomato production.In the case of V-PH production, the presentation of energy and material balances was omitted due to agreements regarding confidential data disclosure.Additionally, emissions occurring on-farm, notably those arising from fertilizer application, were computed utilizing the data outlined in Tables 6 and 7.

Emissions Coefficient
kg N 2 O-N to kg N 2 O [28,44] Table 7. Coefficients for calculating on-farm emissions related to the application of fertilisers for tomato production.

Coefficient Emission Fate
Emissions from mineral fertilizers [45] N in mineral fertilizer 0.01 N 2 O-N to air Indirect N 2 O from atmospheric deposition of fertilizers [45] N in mineral fertilizer 0.01 × 0.1 N 2 O-N to air Subsequently, the outcomes of these computations were consolidated and presented in Table 8.Furthermore, the inventory encompasses the process of biostimulant production (plant stimulating peptides), specifically sourced from soybeans.

Life Cycle Assessment and Carbon Footprint
Life Cycle Assessment and Carbon Footprint was applied only to greenhouse tomato production.
The LCA model was developed in SimaPro software (version 9.5.0.0).In this study, the ReCiPe 2016 midpoint v1.03 method converted the data inventory into conversational indicators [40].In order to comprehend the significance of indicators, the environmental results were normalized.Therefore, among a total of eighteen impact categories assessed, the following eleven categories with significant effects have been identified: Global warming, Ozone formation-human health, Ozone formation-terrestrial ecosystems, Freshwater eutrophication, Terrestrial ecotoxicity, Freshwater ecotoxicity, Marine ecotoxicity, Human carcinogenic toxicity, Human non-carcinogenic toxicity, Fossil resource scarcity, and Water consumption.Global warming, as measured using the IPCC methodology, has been assessed over a span of 100 years.The concept of global warming potential quantifies the extra radiative forces accumulated over a century due to the emission of 1 kg of greenhouse gas compared to the emission of the same mass of CO 2 over the same period.A large set of greenhouse gas emissions (207 GHGs in total) is involved in measuring global warming potential.This comprises a range of gases such as carbon dioxide, methane, nitrogen oxide, chlorofuorocarbons, hydrochlorofluoro carbons, hydrofluorocarbons, chlorocarbons and hydrochlorocarbons, bromocarbons, hyrdobromocarbons and halons, fully fluorinated species and halogenated alcohols and ethers.The global warming potentials (kg CO 2 eq per kg greenhouse gases) are presented in Table A1 in Appendix A.

Partial Budget Analysis
In line with the procedure previously described by Djidonou et al. [46], a partial budget analysis was performed to assess the cost-effectiveness of replacing standard NPK fertilizers (Cerbero ® ) with the NPK fertilizers enriched with plant stimulating peptides (Cerbero Green ® ).Compared to control conditions (fertilization program based on the use of Cerbero ® ), the gross added yield and added costs of using the fertilization program with Cerbero Green ® were calculated and, from these, the net added yield was calculated by difference.

Statistical Analysis
All data were subjected to ANOVA using the SPSS22 software package (Chicago, IL, USA).Means were separated using Tukey's range test performed at 5% level of significance.

Conclusions
The growing need to increase agricultural production to support a continuously expanding population has prompted the scientific community to propose alternative and sustainable production technologies.In this context, biostimulants have played and are playing a key role.In addition to reducing the incidence of abiotic stress, an increasing number of studies have begun to assess the possibility of reducing the use of chemical inputs such as mineral fertilizers by using natural products like biostimulants.The results of our experiment confirm how the use of NPK fertilizers enriched with V-PH (Cerbero Green ® ) improves the productive performance of soilless-grown tomatoes.Specifically, the results have shown that the use of Cerbero Green ® leads to higher marketable yields while reducing fertilizer usage and simultaneously ensuring a positive net change in profit for the grower.In addition, the carbon footprint estimation results revealed that using Cerbero Green ® could reduce the global warming potential of greenhouse-grown tomatoes by 5-8%.This positive outcome was primarily attributed to the increased productivity of the crops.Additionally, when considering other impact categories, the use of Cerbero Green ® demonstrated reductions of 4-11% and 5-9% in standard and decreased fertilization scenarios, respectively.These findings offer valuable insights into the sustainable management of vegetable crops, especially regarding the effective utilization of vegetal-derived protein hydrolysates containing plant stimulating peptides as additives of mineral fertilizers.

Figure
Figure 1.Process contribution to global warming of greenhouse tomato under four treatments per functional unit.

Table 1 .
Effect of fertilization type and rate on marketable fruit yield and yield components of soilless tomato.

Table 2 .
Comparative environmental results of greenhouse tomato under different fertilizer treatments (ReCipe 2016, FU: 1 t).

Table 2 .
Comparative environmental results of greenhouse tomato under different fertilizer treatments (ReCipe 2016, FU: 1 t).

1 .
Process contribution to global warming of greenhouse tomato under four treatments per functional unit.

Table 3 .
Additional revenue and variable costs, and net change in grower profit resulting from the replacement of Cerbero ® by Cerbero Green ® fertilizer at the same fertilization rate.
Table 4presents a between the LCA results of our current study and several other LCA studies conducted on tomato production in different European countries. comparison

Table 4 .
Comparison of greenhouse gas emission of the current study with some existing studies in other European countries.

Table 5 .
Full fertilization plan (named 100%) used in the tomato production.

Table 6 .
Coefficients for conversion of emissions.

Table 8 .
Inventory data for tomato production in relation to fertilizer treatments.