ES3031959A1 - PROCEDURE FOR THE SUITABILITY OF A CORN MACERATION LIQUOR FILTER CAKE AND ITS USE - Google Patents

Abstract

Process for the adaptation and use of a corn steep liquor filter cake. The present invention relates to a process for treating a corn steep liquor filter cake to produce high-value organic matter useful as a biostimulant for plants. The process comprises adding a preservative to a corn steep liquor filter cake and homogenizing the mixture to properly mix it. A drying and grinding process is carried out to produce a solid corn steep liquor cake with biostimulant activity. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Procedure for the adaptation of a corn steep liquor filter cake and its use

The present invention relates to a process for adapting a corn steep liquor filter cake to produce a high added value organic matter for biostimulant use in plants.

Therefore, the present invention could be framed in the field of agriculture and, more specifically, in the production of plant biostimulants.

BACKGROUND OF THE INVENTION

Corn steep liquor (CSL) (CAS 66071-94-1) is the first by-product of the corn wet milling process. In this process, husked, air-cleaned corn is soaked or steeped at 45 to 50°C for 30 to 48 hours in water initially containing approximately 0.1 to 0.2% sulfur dioxide. During the steeping process, soluble components of the corn dissolve in the steep liquor, softening the corn, facilitating further milling.

The steep liquor, sometimes called light steep liquor, is separated from the softened corn and concentrated by evaporation to a solids content of approximately 50%; the resulting concentrate is known in the art as "heavy steep liquor" or more commonly as "corn steep liquor."

This high concentration of solids causes industrial handling problems, as these solids settle and clump together, forming two distinct phases of uneven color: a brown, caked mass at the bottom and a black liquid at the top. This, in turn, makes the product more susceptible to fungal growth on the surface, spoiling the product and diminishing its qualities as a plant fertilizer.

Corn steep liquor contains dissolved minerals, carbohydrates and nitrogenous compounds from corn, as well as organic acids (particularly lactic acid) and modified corn proteins (amino acids and polypeptides) resulting from the enzymatic activity associated with one or more spontaneous fermentations that occur during the corn steeping process.

Therefore, corn steep liquor, due to its high suspended solids content and nutrient richness, suffers from problems in industrial handling such as gel formation, precipitation of suspended solids, and various fungal contaminations. To improve CSL handling, some producers use filtration techniques to separate suspended solids and use only the supernatant, which would be the filtered CSL. Methods such as filter press, nucha filter, or micro/ultrafiltration are commonly used. Filtration processes for CSL are described in patent document CN102660595A, and US8309711B2 shows a filtration process for corn starch sludge. Conditioning corn steep liquor generates a byproduct known as CSL filter cake, which must be managed as waste and incurs additional costs.

Several alternatives have been proposed in the state of the art for using CSL in a solid state, such as European patent application EP1043337A1, which claims a powder-enriched CSL obtained by spray-drying for use in the growth of microorganisms. Japanese patent JPH10338584A describes the production of a solid fertilizer by mixing CSL with dry waste of plant or animal origin and subsequently subjecting said mixture to a drying process. In both cases, CSL is used directly; the prior separation step by filtration, which generates two products: filtered CSL and filter cake, is not performed.

Patent EP1043337A1 uses a spray drying process, which entails very high initial investment costs due to the acquisition of expensive equipment and installation, as well as costly maintenance. Furthermore, this process subjects the CSL to high temperatures, resulting in high energy consumption. Furthermore, spray drying operates at high temperatures, causing the loss of volatile compounds and the inactivation of heat-sensitive enzymes in the CSL. Finally, this process is very difficult to control particle size, and agglomerates or lumps can easily form in the final CSL powder.

Regarding patent JPH10338584A, as in the previous case, CSL is used directly, without a cake separation process, and different materials are used to adsorb CSL. This results in a significantly lower amount of CSL in the final product, as it will be diluted with the adsorbents. Furthermore, many of the adsorbents mentioned are bran or insoluble fibers, so the application of this product will be limited to systems compatible with these types of products, usually top dressing.

Finally, it should be noted that in patent JPH10338584A the product is dried between 60 and 110 degrees Celsius; at 60 degrees the vast majority of enzymes, proteins, vitamins and volatile compounds are already denatured and/or lost.

In view of the aforementioned drawbacks, the present invention proposes a process for obtaining a CSL cake for use as a biostimulant, which maintains its active compounds (without degrading), is highly soluble and can be applied by fertigation, facilitating its handling, increasing the availability and speed of absorption for plants and therefore reducing the application doses.

DESCRIPTION OF THE INVENTION

The conditioning of corn steep liquor generates a byproduct known as CSL filter cake, which must be managed as a waste product and incurs additional costs. However, the inventors of the present invention have demonstrated that this cake, considered in the prior art as a waste product or byproduct, can be used as a powdered plant biostimulant when properly treated or conditioned, as it is very rich in nutrients such as amino acids, proteins, vitamins, and organic acids, among other compounds.

Therefore, in the present invention, a process has been developed that allows the CSL filter cake to be revalued, so that it can be used as a fertilizer or plant biostimulant.

Then, in a first aspect, the present invention relates to a method of treating or adapting a filter cake of corn steep liquor (method of the present invention) to produce a powder cake, where said method comprises the following steps:

a. Stirring a quantity of corn steep liquor filter cake (starting cake) at a speed of between 10 rpm and 1000 rpm and, maintaining these conditions, adding a preservative in an amount of between 0.1% and 1% by weight with respect to the weight of the starting cake,

b. Stir the mixture obtained in (a) at a speed between 10 rpm and 1000 rpm for a time between 1 min and 120 min;

c. drying the mixture obtained in (b) until a product with a moisture content between 0.1% and 10% by weight (% by weight of water) is obtained,

d. grinding the product obtained in (c) to obtain said product in a powdery state (powder cake).

In the present invention, "corn steep liquor" or simply "corn steep liquor" is understood to mean a viscous liquid mixture, a by-product of the corn processing industry, consisting entirely of the water-soluble components of corn steeped in said solvent and whose suspended solids easily tend to agglomerate and precipitate. In addition, it is easily contaminated by fungi and other microorganisms that grow on the surface. This product is commercially available and corresponds to CAS number 66071-94-1.

Corn processing begins with steeping. This steeping of corn grain is accomplished by mixing the corn with water in open tanks at 45 to 52°C for 40 to 48 h. Sulfur dioxide (SO2) is added to prevent fungal growth and help solubilize the material. Initial SO2 concentrations are between 0.1 and 0.2% by weight (pH 3.8 to 4.5) and decrease to 0.05% and 0.01% after 5 and 10 h, respectively.

A natural and spontaneous fermentation occurs in the mash water, where lactic acid bacteria populations increase as SO2 concentrations decrease. This separates the starch from the gluten, solubilises and breaks down the proteins, and softens the corn for easier milling. The corn mash liquor rich in amino acids and peptides is collected and concentrated to obtain a product characterized in that it comprises at least an average of 35-45% by weight of total organic matter where said total organic matter comprises between 25-35% by weight of the total product of fulvic acids, 5-6% by weight of free amino acids, 15-25% by weight of total amino acids, B vitamins 8000-9000 micrograms/kilogram, lactic acid 5-6% by weight and betaine 0.1-0.5% by weight, where it also contains 3-4% by weight of total nitrogen and between 2-4% by weight of total potassium.

In the present invention, "corn steep liquor filter cake" is understood to mean the concentrated product obtained by the elimination or separation of the liquid phase of the corn steep liquor such that its moisture percentage is reduced to 20% by weight or less. It is, therefore, a product of solid or semi-solid consistency. This cake concentrates a large quantity of bioactive components from the CSL and generally contains between 4 - 6% w/w of free amino acids, 15-25% w/w of total amino acids, a crude protein of 25 - 35% w/w, carbohydrates between 3.5 and 5% w/w, of which 3 - 4% w/w are total sugars and crude cellulose between 0.1 - 1% w/w.

The stirring speed in steps a) and b) must be kept within the indicated ranges, since at speeds above 1000 rpm, the product clumps in the mixer and does not homogenize properly. A speed below 10 rpm is not sufficient to properly integrate and dry the product.

In a preferred embodiment, step a) and/or b) are carried out in a Cowles type agitator (a agitator consisting of a toothed disc with inclined radial blades placed at the bottom and top in an alternating manner), in a high shear homogenizer (the machine that shears, disperses and impacts the material through the high speed rotation of the homogeneous head connected to the motor), in a paddle mixer, in a kneading mixer or in an agitator with special blades for high viscosity products, these devices being well known in the state of the art to those skilled in the art.

In a preferred embodiment, between 500 kg and 1000 kg of starting corn steep liquor filter cake are used in step a).

In a preferred embodiment, the stirring time of step b) is at least 15 min, more preferably, 15 min.

In a preferred embodiment, the added preservative is selected from the following: citric acid, potassium sorbate, octylisothiazolinone (OIT), (ethylenedioxy)dimethanol (EDDM), iodopropynyl butylcarbamate, phenoxyethanol, benzoisothiazolinone, 2-(thiocyanomethylthio)-benzothiazole, orthophenylphenol, parachlorometacresol, methylchloroisothiazolinone, methylisothiazolinone, glutaraldehyde, and any combination thereof. In a more preferred embodiment, the preservative is octylisothiazolinone, (ethylenedioxy)dimethanol, or a combination thereof. More preferably, the added preservative is a 1:1 mixture by weight of octylisothiazolinone and (ethylenedioxy)dimethanol.

In a preferred embodiment, the drying of step c) is carried out in a vacuum paddle dryer, preferably working between 30 to 50°C, more preferably 45°C, and at a pressure between -1013 mbar and 2026 mbar, more preferably, 50 mbar.

To verify the degree of humidity obtained in step c), periodic samples are taken and an analytical control is carried out using Karl-Fischer titration or loss on drying balance.

In another preferred embodiment, drying is carried out by adding a water-absorbent or hygroscopic compound to the mixture obtained in step b).

These absorbent compounds, individual or in mixture, can be Lemna sp. and its flours, macroalgae of the genus Ascophyllum sp., Ecklonia sp., Macrocystis sp. or Laminaria sp., cellulose, polyacrylates, diatomaceous earth, expanded or unexpanded perlite, vermiculite, maltodextrins, silica and calcined or uncalcined kaolin. They are added in an amount of between 10% and 90% of the absorbent compound or compounds with respect to the cake obtained in b). Preferably, between 20% and 50% by weight and, even more preferably, 50% by weight are added.

In a preferred embodiment, the grinding in step d) is carried out using a micronizing mill. Preferably, a stainless steel micronizing mill with a pulverizing disc and a sieve for classifying particles by size. A 100-micron sieve is generally used, but the essential requirement is that the material remains in powder form and free of lumps, regardless of the particle size.

In a preferred embodiment, in step c), after drying, a powdered antifoam based on a combination of polyether polyols and amorphous silica is added to the mixture at a rate of between 0.1% and 2% by weight relative to the dried cake obtained in c), preferably 1% by weight. The antifoam prevents the formation of foams in agricultural application systems; these foams can be so dense that they can impede application of the product and leak out of the application tanks.

The advantage of the process of the invention, in addition to the utilization or revaluation of the corn steep liquor filter cake for use as a biostimulant, is the extension of its shelf life, which is at least two years. Furthermore, the cake obtained, which has a very low moisture content, concentrates all the active ingredients without them degrading, is easy to store, and is cheaper to distribute because it does not require transporting an excessively bulky product. Additionally, it is a homogeneous, powdery dry product, so it will solubilize in the agricultural application broth, avoiding blockages in dosing facilities and equipment and distributing the compounds more evenly. Likewise, as demonstrated in the examples of the present invention, the cake treated by the process of the present invention has a greater biostimulant effect than the untreated or conditioned cake and the corn steep liquor itself (see example 4 in the examples section).

In the process of the present invention, any cake resulting from the elimination or separation of the liquid phase of the corn steep liquor to obtain a moisture content of 20% by weight or less, as defined above, can be used as the starting cake. However, in a preferred embodiment, the starting cake used in the present invention is obtained by a filter-pressing process of the corn steep liquor comprising the following steps:

- a solution of perlite between 0.1% and 5% by weight is made in water with constant stirring,

- the previous solution is passed through a filter press to form a pre-layer of perlite on the filter itself, (the water passes through the filter, while the perlite does not, thus leaving a layer of perlite on the filter),

- on the other hand, between 0.1% and 5% by weight of perlite is added to the corn liquor with constant stirring (the % by weight indicated is with respect to the liquor from which we start); in a preferred embodiment, prior to adding perlite to the liquor, the corn liquor is kept stirring in air for between 1 and 30 days, since this oxygenates the mixture and induces spontaneous oxidation and hydrolysis processes that improve filtration;

- the corn liquor is filtered by applying a pressure of between 1 and 15 bars, preferably plus 10 bars;

- Finally, the cake retained on the filter plates of the filter press is recovered and this cake is used as the starting material for the process of the invention.

For example, to form the perlite pre-layer, 1000 to 1500 liters of solution are passed through a filter press with 470 mm diameter filters.

Given the characteristics of corn steep liquor and its consistency, filter-pressing is not possible without the addition of perlite, which forms a pre-layer of this compound on the filter. Perlite prevents the product from caking, making it easier to work with.

Furthermore, the presence of perlite in the product offers several advantages. The first is that the final product does not clump because it acts as a solids dispersing agent, prevents hygroscopicity of the dry cake, and also acts as a soil aerator, improving its quality. Furthermore, the adsorbent capacity of expanded perlite allows it to accumulate certain liquor compounds in its matrix and slowly release them.

A second aspect of the invention relates to a filter cake conditioned or treated according to the process described in the first aspect of the invention. The cake obtained is in a powdery state and has a moisture (water) content of less than 10% by weight and a preservative content of between 0.1 and 2% by weight.

In a preferred embodiment, the conditioned or treated filter cake comprises perlite in an amount between 0.5% and 5% by weight.

In a preferred embodiment, the conditioned or treated filter cake has an antifoam content of between 0.1% and 3% by weight.

In a preferred embodiment, the conditioned or treated filter cake contains between 4 - 8% by weight of free amino acids, 15-30% by weight of total amino acids, a crude protein of 25 - 40% by weight, carbohydrates between 3.5 and 6% by weight of which 3 - 5% by weight are total sugars and crude cellulose between 0.1 - 2% by weight.

A third aspect of the present invention relates to the use of the powdered corn steep liquor cake obtained by the process of the present invention as a plant biostimulant.

From the experimental results obtained (see example 4 of the present invention), it is observed that under conditions of saline stress, plants treated with conditioned cake, in general, obtain significant improvements with respect to those treated with unconditioned cake.

Specifically, in biomass production parameters, the conditioned cake obtained significant increases in fresh and dry weight of the shoot and in leaf area compared to plants treated with unconditioned cake. Furthermore, oxidative stress indicators show that plants treated with conditioned cake have significantly lower levels of malondialdehyde (MDA) and reactive oxygen species (O2 and H2O2) and are therefore less stressed than plants treated with unconditioned cake. Similarly, plants treated with conditioned cake show greater production of antioxidant compounds such as phenols, ascorbate, and glutathione and, therefore, greater FRAP and TEAC antioxidant capacity.

Regarding photochemical activity and vitality, plants treated with conditioned cake showed higher values in the main indices (Fv/Fm; RC/ABS and PIabs) compared to plants treated with unconditioned cake.

Continuing with photosynthesis efficiency, plants treated with conditioned cake obtain an improvement in transpiration rate (E), stomatal resistance (r), water use efficiency (WUE) and net photosynthetic rate (A) while plants treated with unconditioned cake obtain worse results.

Finally, it is observed that plants treated with conditioned cake have a lower foliar concentration of toxic ions (Na+ and Cl) than plants treated with unconditioned cake and, on the contrary, plants treated with conditioned cake obtain increased values of beneficial cations (K+) while untreated plants have a much lower concentration of these cations.

In another experiment carried out, it is deduced that under stress conditions due to limited nitrogen fertilization, it is observed that, in general, plants treated with conditioned cake obtain significantly better results in the main indicators than plants treated with corn steep liquor (CSL-B).

Under conditions of moderate (N-50%) and severe (N-25%) nitrogen fertilization limitation, an increase in the production of aerial biomass is observed in plants treated with conditioned cake, both in fresh and dry weight and in leaf area with respect to plants treated with CSL-B.

In terms of N assimilation parameters, under all nitrogen fertilization conditions, plants treated with conditioned cake show greater glutamine synthetase (GS) activity than plants treated with CSL-B. Furthermore, under severe nitrogen limitation (N-25%), plants treated with conditioned cake also show increased nitrate reductase (NR) levels.

Without nitrogen limitation (N-100%), plants treated with conditioned cake showed significantly lower foliar concentrations of potentially toxic nitrogen forms (nitrates) than plants treated with CSL-B. On the other hand, under moderate (N-50%) and severe (N-25%) nitrogen limitation conditions, plants treated with conditioned cake showed an increase in foliar organic nitrogen (a nutritionally beneficial form of nitrogen), while plants treated with CSL-B had a lower concentration.

Under N-limiting conditions in the growing medium N-50% and N-25%, the application of conditioned cake significantly improved nitrogen use efficiency (NUE) and nitrogen utilization efficiency (NUtE).

In conclusion, it can be indicated that the conditioned cake has a greater and novel performance as a biostimulant compared to the unconditioned cake and corn steep liquor.

"Plant biostimulant" means a product whose function is to stimulate plant nutrition processes, regardless of the nutrient content of the product, with the sole objective of improving one or more of the following characteristics of plants and their rhizosphere:

a) efficiency in the use of nutrients,

b) tolerance to abiotic stress,

c) quality characteristics, or

d) availability of nutrients immobilized in the soil and rhizosphere.”

The application rate of treated or conditioned cake for use as a biostimulant is between 1 and 10 kg/hectare. This is a much lower dose than that required for other biostimulants known in the art, which is typically between 100 and 10,000 kg/hectare.

The conditioned liquor cake preserves the bioactive substances that exert a biostimulating effect on plants for a long time (at least 2 years) without them degrading, unlike what happens with untreated or conditioned cake, so its use is prolonged.

A fourth aspect of the present invention relates to a fertilizer characterized in that it comprises the biostimulant corn steep liquor cake obtained by the process of the present invention.

Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

Example 1: Obtaining the starting wet cake of the process of the present invention from corn maceration liquor by means of a pressed filter process

In a preferred embodiment, the starting cake used in the present invention is obtained by means of a filtration system consisting of a plate filter press from the company Grupo Tecnológico Mediterraneo, S.L. (it is a pressure filtration system) of the corn maceration liquor comprising the following stages:

- a solution of perlite (CAS 93763-70-3) at 1% by weight in water is made with constant stirring;

- 1000L of the above solution are passed through the filter press to form a perlite pre-layer.

On the other hand, 1% by weight of perlite is added to the corn steep liquor (CAS 66071-94-1) under constant stirring;

- The corn liquor is filtered through the filter press by applying 10 bars of pressure to the filter press;

- Finally, the cake retained in the filter is blown with compressed air, which allows the filter to be cleaned and the wet cake to be recovered without dilution, since no additional water is added.

Example 2: Example of carrying out the process of the present invention starting from the cake obtained in Example 1

Starting from 100kg of wet cake obtained in example 1, the following steps are carried out:

a. Stir 100 kg of wet corn steep liquor filter cake (starting cake) at a speed of 300 rpm and, maintaining these conditions, add a mixture (1:1 by weight) of octylisothiazolinone (OIT) and (ethylenedioxy)dimethanol (EDDM) in the amount of 0.3 kg,

b. Stir the mixture obtained in (a) at a speed of between 300 rpm for a time of between 30 min;

c. drying the mixture obtained in (b) in a vacuum paddle dryer at 45°C and a pressure of 50 mbar, controlling the humidity using the Karl Fischer method until a product with a humidity of 10% by weight (% by weight of water) is obtained,

d. add 1 kg of powdered antifoam based on polyether polyols and amorphous silica (DENSIPOL PW 1, purchased from Chemipol) to the dryer, mix by moving the dryer for 10 min; e. grind the product obtained in (d) using a micronizing mill to obtain said product in a powdered state (dry powder cake).

Example 3: Cake characterization

After analyzing the cake obtained in example 1, the following data were obtained: Humidity 10% by weight, a preservative content of 0.3% by weight, and an antifoam content of between 0.1% and 3% by weight. It contains between 5% w/w of free amino acids, 25% w/w of total amino acids, a crude protein of 33% w/w, carbohydrates 4.8% w/w, of which 3.8% w/w are total sugars, and crude cellulose 0.8% w/w.

The measurement techniques used are as follows: Free and total amino acids: HPLC - fluorescence chromatography; crude protein: Kjeldahl distillation and calculation; carbohydrates: polarimetry and Luff-Schoorl titration; total sugars: Luff-Schoorl titration; and crude cellulose: gravimetry.

Example 4: Application of the cake obtained in example 2 as a plant biostimulant

4.1. Plant material and growth conditions

Pepper plants (Capsicum annuumcv. Alycum) were used to conduct the experiments in this example. Seeds from these plants germinated and grew for 45 days in a cell tray (cell size, 3 cm x 3 cm x 10 cm) at the Saliplant S.L. Seedling Farm (Carchuna, Granada). Seedlings were then transferred to a growth chamber at the Department of Plant Physiology, University of Granada, under controlled conditions of 60–80% relative humidity, 29°C/20°C (day/night), and 16 h/8 h photoperiod with a PPFD (photosynthetic photon-flux density) of 450 ^m or l'V 1 (measured with an SB quantum 190 sensor, LI-COR Inc., Lincoln, NE, USA).

Under these conditions, plants were grown in individual pots (13 cm top diameter, 10 cm bottom diameter, 12.5 cm high, and a volume of 2 L) filled with a perlite:peat mixture. Fertilization consisted of a complete Hoagland-type nutrient solution composed of: 4 mM KNO3, 2 mM Ca(NO3)2, 2 mM MgSO4, 1 mM KH2PO4, 1 mM NaH2PO4, 2 |<j>M MnCb, 1 |JM ZnSO4, 0.25 |<j>M CuSO4, 0.1<j>M Na2MoO4, 125<j>M Fe-EDDHA, and 50<j>M H3BO3, with a pH of 5.8.

4.2. Experimental design and plant sampling

4.2.1. Salinity Experiment

After 49 days of germination, the treatments shown in the following table were applied:

The application of the different treatments began 7 days after the plants were transplanted into the growth chamber. The Torta_A (conditioned according to the present invention) and Torta_NoA (dry only, unconditioned) products were applied to the roots four times, with a frequency of 7 days between each application. Finally, the experimental design consisted of a complete randomized block with eight plants per treatment arranged in individual pots, with the treatments randomly distributed throughout the growth chamber.

Seven days after the last treatment application, all plants were immediately processed for further analysis. The plant material was decontaminated and subsequently dried on filter paper to obtain fresh weight (FW). Half of the samples, either fresh or frozen at -40°C, were used for the analysis of the following parameters: leaf area, chlorophyll a fluorescence (Fv/Fm, RC/ABS, PI(Abs) and 1 -Vj), photosynthetic efficiency (Infra-Red Gas Analyzer (IRGA-LiCOR 6400), malondialdehyde (MDA) and oxygenated free radicals (ROS: O2 " and H2O2) concentrations, phenol, ascorbate, glutathione and proline concentrations, and FRAP and TEAC antioxidant tests. The other half of the plant material, after drying in a forced air oven, was used to determine the dry weight (DW) and the concentrations of Na, Cl and K ions.

4.2.2. Experiment Limitation in nitrogen fertilization

After 49 days of germination, the treatments shown in the following table were applied:

*N-100% means that the fertigation nutrient solution has 8mM of nitrogen. This is the full recommended dose to ensure that crops do not suffer from nitrogen deficiency.

N-50% indicates that the nutrient solution has 4mM of nitrogen. That is, half of the recommended dose, and therefore, the crop will develop a moderate deficiency due to nitrogen deficiency. N-25% is the same as above but with 2mM of nitrogen to induce a severe deficiency. T4, T5, and T6 would be the same as above but supplemented with Corn Steep Liquor (CSL-B) to determine the biostimulant effect in each nitrogen fertilization situation.

T7, T8, T9 are the same as above, but supplemented with Conditioned Cake. This allows for a comparison of the effects of CSL-B and Conditioned Cake.

The application of the different treatments began 7 days after the plants were transplanted into the growth chamber and the CSL-B products (corn steep liquor and Torta_A) were applied to the roots 4 times with a frequency of 7 days between each application according to the recommendation of the R&D team of the company Atlántica Agrícola S.A. Finally, the experimental design consisted of a complete random block with 8 plants per treatment arranged in individual pots, with the treatments randomly distributed in the growth chamber.

Seven days after the last treatment application, all plants were immediately processed for analysis. The plant material was decontaminated and subsequently dried on filter paper to obtain fresh weight (FW). Half of the samples, either fresh or frozen at -40°C, were used for analysis of the following parameters: leaf area, nitrogen metabolism enzyme activity (nitrate reductase, NR; and glutamine synthetase, GS), and soluble amino acid and protein concentrations. The other half of the plant material, after drying in a forced-air oven, was used to determine dry weight (DW), as well as the concentrations of different forms of N (total N, organic N, and nitrates) and the N-use efficiency (NUE) formulas.

4.3. Analysis of Plant Material

4.3.1. Leaf area

The leaf area was measured using a LI-COR optical reader, model LI-3000A.

4.3.2. Analysis of chlorophyll a fluorescence

Plants were dark-adapted for 30 min before measurements were taken using a special leaf clip placed on each leaf. Chl a fluorescence kinetics were determined using the Handy PEA Chlorophyll Fluorimeter (Hansatech Ltd., King’s Lynn, Norfolk, UK); OJIP phases were induced by red light (650 nm) at a light intensity of 3000 ^mol photons m"2 s-1. The OJIP fluorescence phases were analyzed by the JIP test (Strasser R, Srivastava A, Tsimilli-Michael M 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In M. Yunus, U. Pathre, P. Mohanty, eds. Probing Photosynthesis: Mechanism, Regulation and Adaptation. London: Taylor & Francis, 443 480.). Measurements were made on fully developed leaves at the mid-plant position. The following parameters obtained from the JIP test were used to study energy fluxes and photosynthetic activity: initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv= Fm-Fo), fluorescence value at 300 ^s (Peak K), maximum quantum product of primary photochemistry (0Po= Fv/Fm), function index (PIabs), proportion of active reaction centers (RCs) (RC/ABS), and the 1-Vj value indicates the electron output primarily from photosystem II (Strasser R, Srivastava A, Tsimilli-Michael M 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In M. Yunus, U. Pathre, P. Mohanty, eds. Probing Photosynthesis: Mechanism, Regulation and Adaptation. London: Taylor & Francis, 443-480.).

4.3.3. Analysis of photosynthetic efficiency

Measurements were recorded using a LICOR 6800 Portable Photosynthesis System infrared gas analyzer (IRGA: LICOR Inc. Nebraska, USA). Intermediate leaves were placed in the measuring cuvettes under optimal growth conditions. Before use, the instrument was warmed up for 30 min and calibrated. Measurements used standard optimal cuvette conditions at 500 ^mol m2 s-1 photosynthetically active radiation (PAR), 400 ^mol mol_1 CO2 concentration, leaf temperature at 30 °C, and 60% relative humidity. The net photosynthetic rate, transpiration rate, and stomatal resistance were recorded simultaneously. Data were stored on the LICOR device and analyzed using the "Photosyn Assistant" software. The instantaneous water use efficiency (WUE) was calculated by dividing the net photosynthesis rate (A) by the corresponding transpiration rate (E) (Strasser R, Srivastava A, Tsimilli-Michael M 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In M. Yunus, U. Pathre, P. Mohanty, eds. Probing Photosynthesis: Mechanism, Regulation and Adaptation. London: Taylor & Francis, 443-480.).

4.3.4. Determination of the concentration of oxidative indicators (MDA, H2O2 and O2-)

For malondialdehyde (MDA) extraction, fresh plant material was homogenized with 5 ml of 50 mM buffer (0.07% NaH2PO4. 2 H2O and 1.6% Na2HPO4. 12 H2O) in a mortar and subsequently centrifuged at 20,000g for 25 minutes in a refrigerated centrifuge. Subsequently, a 1 ml aliquot of supernatant was mixed in test tubes with 4 ml of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The resulting mixture was heated at 95°C for 30 minutes and then rapidly cooled in an ice bath. The samples were then centrifuged at 10,000g for 10 minutes and the absorbance of the supernatant was measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted from the reading obtained at 532 nm. The MDA concentration was calculated using the molar extinction coefficient of MDA of 155 mM-1cm-1 (Fu J, Huang B 2001. Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress. Env. Exp. Bot.

45: 105-114.).

The H2O2 concentration was measured colorimetrically according to Mukherjee and Choudhuri (Mukherje SP, Choudhuri MA 1983. Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol. Plant. 58: 166-170.). Fresh plant material was homogenized in cold acetone. A 1 mL aliquot of the extract was mixed with 200 ^L of 0.1% titanium dioxide in 20% (v:v) H2SO4 and the mixture was centrifuged at 6000g for 15 minutes. The intensity of the yellow color of the supernatant was measured at 415 nm. The H2O2 concentration was calculated from a H2O2 standard curve.

The O2- concentration was measured colorimetrically according to Barrameda-Medina et al. (Barrameda-Medina Y, Montesinos-Pereira D, Romero L, Blasco B, Ruiz JM 2014. Role of GSH homeostasis under Zn toxicity in plants with different Zn tolerance. Plant Sci.

227: 110-121.). 0.1 g of plant material was macerated and 300 ^L of 50 mM phosphate buffer was added, and the mixture was centrifuged at 10,000 g for 15 minutes. From the supernatant, 250 ^L were taken, to which 50 mM phosphate buffer and 250 ^L of 10 mM hydroxylamine were added, and the mixture was incubated for 20 min at 25 ° C. Subsequently, 60 ^L were taken from the supernatant and 180 ^L of 17 mM sulfonylic acid and 180 ^L of 7 mM α-1-naphthylamine were added, and the mixture was incubated for 1 hour at room temperature. After the incubation time, the color intensity was measured at 530 nm. The O2 concentration was calculated from a standard O2 curve.

4.3.5. Determination of the concentration of the compounds: total phenols, ascorbate, glutathione and proline

Total phenols in plant tissue were extracted with methanol. The content was quantified at an absorbance of 765 nm using the Folin-Ciocalteau reagent (Rivero RM, Ruíz JM, García PC, López-Lefebre LR, Sánchez E, Romero L 2001 Resistance to cold and heat stress: accumulation of phenolic compounds in tomato and watermelon plants. Plant Sci. 160: 315-321.). The phenol concentration was obtained using a caffeic acid standard curve.

For the extraction and quantification of ascorbate (AsA) the Law method was used (Law MY, Charles SA, Halliwell B 1992. Glutathione and ascorbic acid in spinach (Spinacea oleracea) chloroplast. The effect of hydrogen peroxide and paraquat. Bichem. J. 210: 899-903.). This method is based on the reduction of Fe3+ to Fe2+ by AsA in acid solution. 0.5 g of frozen plant material was homogenized in 5 ml of 5% (w/v) metaphosphoric acid and subsequently centrifuged at 16000 g at 4oC for 15 min. Then, 0.2 ml of supernatant was added to a test tube together with 0.5 ml of 150 mM sodium phosphate buffer (pH 7.5) and 0.1 ml of distilled H2O. The mixture was shaken and incubated at room temperature in the dark for 10 min. Next, 0.1 ml of 0.5% (w/v) N-ethylmaleimide, 0.4 ml of 44% (v/v) orthophosphoric acid, 0.4 ml of 4% (w/v) 2,2'-bipyridyl in 70% ethanol and 0.2 ml of 3% (w/v) FeCh were added. The test tubes were then shaken and incubated at 40 °C in the dark for 40 min. Finally, the absorbance at 525 nm was measured against an AsA standard curve.

GSH concentration was determined using the Law method (Law MY, Charles SA, Halliwell B 1992. Glutathione and ascorbic acid in spinach (Spinacea oleracea) chloroplast. The effect of hydrogen peroxide and paraquat. Bichem. J. 210: 899-903.). This method is based on the specificity of the GSH reductase (GR) enzyme for oxidized glutathione (GSSG). Extraction was first carried out by homogenizing 0.5 g of fresh material with 5 mL of 5% (v/v) metaphosphoric acid. The homogenate was filtered and centrifuged at 16,000 g for 15 minutes at 0°C. For the quantification of total GSH, a reaction mixture containing 50<j>L of extract, 250<j>L of 50 mM Heppes-HCl buffer (pH 7.6) containing 330 mM betaine, and 150<j>L of 10% (v/v) sulfosalicylic acid was prepared. Subsequently, 150<j>L of the previous reaction mixture, 700 |<j>L of 0.3 mM NADPH and 100<j>L of 6 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were added to a test tube. The mixture was stirred and after 4 minutes of waiting, 50<j>L of GR (10 U/mL) was added. Finally, the samples were read at 412 nm against a GSH standard curve.

For the determination of the free proline concentration, the leaves were homogenized in 5 ml of 96% ethanol. The insoluble fraction of the extract was washed with 5 ml of 70% ethanol. The extract was centrifuged at 3500g for 10 minutes and the supernatant was stored at 4°C for the determination of proline according to the method described by (Irigoyen JJ, Emerich DW, Sánchez-Díaz M 1992. Water stress induced changes in the concentrations of proline and total soluble sugars in nodulated alfafa (Medicago sativa) plants. Physiol. Plant. 84: 55-60.).

4.3.6 FRAP and TEAC antioxidant tests

The FRAP assay was performed with FRAP reagent, composed of 1 mM 2,4,6-tripyryldyl-2-triazine (TPTZ) and 20 mM FeCh in 0.25 M CH3COONa, pH 3.6. A 100 μl extract obtained from leaf homogenization in 10 ml of methanol was added to 2 ml of FRAP reagent. The mixture was then incubated at room temperature (20°C) for 5 min. The absorbance was measured at A593 against a standard curve of 25-1600 μM Fe3+ prepared using a 25 mM ferrous sulfate stock solution (Benzie IEF, Strain JJ 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Ann. Biochem. 239: 70-76.).

The TEAC (Trolox Equivalent Antioxidant Activity) test was carried out using a modified version of the method of (Cai Y, Luo M, Sun HC 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medical plants associated with anticancer. Life Sci. 74: 2157–2184.). First, 7 mM of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was mixed with 2.45 mM of potassium persulfate to produce the ABTS+ cation, and the resulting mixture was incubated for 16 h in the dark at room temperature. The resulting ABTS solution was then diluted with methanol, and its absorbance was adjusted (using methanol as a blank) until it recorded a value of 0.7 ± 0.02 at a wavelength of 734 nm. A 100 ^L aliquot of leaf extract (0.5 g/10 mL of methanol) was mixed vigorously with 3.9 mL of diluted ABTS solution, and left in the dark at room temperature for 6 minutes, after which the absorbance at 734 nm was recorded. The samples were then challenged with a 0-15 ^M trolox standard curve following the same procedure as above.

4.3.7. Enzymatic Activities of Nitrogen Metabolism

Nitrate reductase (NR) activity was determined following the procedure described by (Navarro-León E, Barrameda-Medina Y, Lentini M, Esposito S, Ruiz JM, Blasco B 2016. Comparative study of Zn deficiency in L. sativa and B. oleracea plants: NH4+ assimilation and nitrogen derived protective compounds. Plant Sci. 248: 8-16). 0.2 g of fresh plant material was macerated in a mortar with 1 ml of extraction buffer containing 2 mM EDTA-Na, 2 mM DTT, 1% (w/v) PVPP in 100 mM KH2PO4 (pH 7.5). The suspension was centrifuged for 20 min at 20,600 g at 4°C. The obtained supernatant was added to the reaction mixture containing: 100 mM KNO3, 2 mM NADH, 10 mM cysteine and 10 mM MgCh in 100 mM KH2PO4 buffer (pH 7.5) and this mixture was incubated at 30°C for 30 min. Subsequently, 1 mM Zn acetate was used as a stop reagent and 1% sulfanilamide in 1.5 M HCl and 0.02% (w/v) NNEDA in 0.2 M HCl for the detection of the formed NO2". Finally, the NR activity was determined by measuring the absorbance of the produced NO2" at 540 nm.

Glutamine synthetase (GS) activity was determined by an adaptation of the hydroxamate synthase assay published by (Navarro-León E, Barrameda-Medina Y, Lentini M, Esposito S, Ruiz JM, Blasco B 2016. Comparative study of Zn deficiency in L. sativa and B. oleracea plants: NH4+ assimilation and nitrogen derived protective compounds. Plant Sci. 248: 8-16.). A total of 0.1 g of leaves was macerated in a mortar with 1 ml of extraction buffer containing: 100 mM sucrose, 2% (v/v) p-mercaptoethanol and 20% (v/v) ethylene glycol in 100 mM maleic acid-KOH (pH 6.8). The suspension was centrifuged for 20 min at 20600g and 4°C. The resulting extract was used to measure GS activity. The reaction mixture used consisted of 150 mM sodium glutamate, 30 mM hydroxylamide and 10 mM ATP as substrates together with 45 mM gS O ^ H2O, and 4 mM EDTA-Na, all dissolved in 150 mM imidazole-HCl buffer (pH 7.8). After incubation at 28°C for 30 min, the formation of glutamylhydroxamate was determined by measuring its absorbance at 540 nm after binding to acidified ferric chloride.

4.3.8. Concentration of amino acids and soluble proteins

For the determination of amino acids and soluble proteins, approximately 0.5 g of plant material was weighed and homogenized with 5 ml of 50 mM phosphate buffer pH 7.0. The homogenate was filtered through 4 layers of gauze and subsequently centrifuged at 12360 g for 15 min. The supernatant was used for the quantification of amino acids and soluble proteins. The concentration of soluble amino acids was quantified by the ninhydrin method (Yemm EW, Cocking EC 1955. The determination of aminoacids with ninhydrin. Analyst 80: 209-213.). Regarding soluble proteins, 0.9 ml of 50 mM phosphate buffer pH 7.0 and 5 ml of Coomassie blue were added to a volume of 0.1 ml of the supernatant. After 20 min, the samples were measured at a wavelength of 595 nm, against a standard curve of albumin (Navarro-León E, Barrameda-Medina Y, Lentini M, Esposito S, Ruiz JM, Blasco B 2016. Comparative study of Zn deficiency in L. sativa and B. oleracea plants: NH4+ assimilation and nitrogen derived protective compounds. Plant Sci. 248: 8-16.).

4.3.9. Concentration of Na, K, Cl, total N, organic N, nitrates, and nitrogen use efficiency

The concentrations of the elements Na, Cl and K were determined by ICP-OES. Leaf samples were subjected to a mineralization process following the Wolf method (Wolf B 1982. A comprehensive system of leaf analyses and its use for diagnosing crop nutrient status. Commun. Soil Sci. Plant Anal. 13: 1035-1059.). A total of 0.2 g of dried leaves were digested with 30% HNO and H2O at 300°C, and the resulting mineralized material was used for the analysis of the ionic elements Na, Cl and K.

For the determination of total N, 0.2 g of dried leaves were ground and mineralized with 98% H2SO4 and 30% H2O2, at a temperature of 300 °C and the mineralized was used for N analysis. The concentration of total N was determined by colorimetry based on the Berthelot reaction, according to the method described by (Krom MD 1980. Spectrophotometric determination of ammonia: a study of a modified Berthelot reaction using salicylate and dichloroisocyanurate. Analysis. 105: 305-316.). For the determination of the concentration of soluble NO3" an aqueous extraction was carried out following the method of (Cataldo DA, Maroon M, Schrader LE, Youngs VL 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6: 71-80.). The determination of NO3" was based on the colorimetric reaction formed by the union of NO3- with salicylic acid in a basic medium (Cataldo DA, Maroon M, Schrader LE, Youngs VL 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6: 71-80.). Organic N was obtained by subtracting total N and soluble nitrates (Wolf B 1982. A comprehensive system of leaf analyzes and its use for diagnosing crop nutrient status. Commun. Soil Sci. Plant Anal. 13: 1035-1059.).

For the calculation of N Utilization Efficiency (NUtE), N Uptake Efficiency (NUpE), N Use Efficiency (NUE), and apparent recovery of N fertilizer (RAN) the equations described by (Xu G, Fan X, Miller AJ 2012. Plant nitrogen assimilation and use efficiency. Annu. Rev. Plant Biol. 63: 153-182.) were used, where NUtE is defined as the quotient between biomass production and the amount of N in the plant, NUpE is defined as the quotient between the amount of N in the plant and the amount of N applied in fertilization, NUE is defined as NUtE x NUpE, and RAN is defined as the quotient between N absorbed in the treated plot minus the N absorbed in the control plot, all of this between the N absorbed in the treated plot.

4.4. Statistical Analysis

The results were evaluated statistically using a simple ANOVA with a 95% confidence interval. Differences between treatment means were compared using Fisher's least significant difference (LSD) test at the 95% probability level. Significance levels were expressed as: * P < 0.05; ** P < 0.01; *** P < 0.001; NS not significant.

4.5. Results and discussion of the salinity experiment

Currently, the main abiotic factors worldwide are water stress and soil salinity. These abiotic stresses affect more than 100 countries and approximately 20% of the world's cultivated land. It is estimated that by 2050, more than 50% of the world's arable land will be affected, causing significant losses in agricultural crop production, especially in arid and semi-arid areas. The impact of these stresses may increase as global climate change progresses. Salt stress causes changes in plant morphological, physiological, and biochemical responses, with a consequent reduction in growth, yield, biomass, and quality. Therefore, this type of stress represents a serious problem for commercial horticulture, as it leads to a loss of productivity, particularly in the Mediterranean region.

The parameters that primarily reliably define the existence of this type of stress are those related to plant growth. In this project, and to test the effect of both conditioned and unconditioned cake on salt stress, we analyzed the following parameters: fresh and dry biomass production of the shoots and leaf area. These parameters reliably indicate plant growth under different growing conditions and, therefore, their ability to adapt to adverse growing conditions.

As can be seen in Table 4.5.1, which reflects the data on biomass production of the aerial part and area, we generally observe that the existence of salinity led to a reduction in growth, with all salinity treatments presenting lower values of biomass production of the aerial part and leaf area than those obtained in the control plants (Table 4.5.1). However, under salinity conditions, the application of cake products, both conditioned and unconditioned, produced a beneficial effect by stimulating the production of fresh and dry biomass of the aerial part, as well as the leaf area with respect to the plants stressed with salinity (Table 4.5.1), with higher increases in fresh biomass production of the aerial part at 37% and in dry biomass production of the aerial part at 25%. It should be noted that the conditioned cake primarily resulted in higher biomass production and leaf area than foliar application (Table 4.5.1). Finally, it should be noted that although the application of the cake product, both conditioned and unconditioned, was beneficial, it did not completely reduce the detrimental effect of salt stress, since biomass production and leaf area values were in all cases lower than those of non-stressed control plants (Table 4.5.1).

Table 4.5.1: Biomass production of the aerial part and leaf area

In short, considering the growth data obtained in this trial, we can conclude:

(i) The cake product exhibits positive biostimulant effects on the growth of pepper plants under salinity stress conditions, with no phytotoxic effects observed in any case. In short, these results clearly indicate a highly significant beneficial effect under salinity conditions on pepper plants to which the cake product was applied, with its application being more effective when conditioned, with statistically significant differences compared to unconditioned application.

(ii) Although the application of the cake product was beneficial, it did not completely reduce the detrimental effect of salt stress on the plants, since growth in all cases was lower than that of non-stressed control plants.

Reducing the accumulation of reactive oxygen species (ROS) is crucial for plant survival under salt stress conditions, so the study of oxidative metabolism has long been used as an indicator of the damage caused by this type of stress. One of the potential protective effects of biostimulants against abiotic stresses is mainly due to the fact that these products, in many plant species, reduce cellular oxidative damage, and therefore membrane lipid peroxidation, by regulating antioxidant defense and decreasing ROS levels in plants. In fact, one of the mechanisms by which biostimulants are thought to improve resistance to abiotic stresses is by inducing both enzymatic and non-enzymatic antioxidant systems in plants.

In this sense, MDA concentration is the indicator parameter of membrane lipid peroxidation, and an increase in its values suggests the excessive presence of ROS. Table 4.5.2 shows that the plants with the highest biomass production in the aerial and foliar parts of this trial indeed showed the lowest MDA levels, i.e., non-stressed control plants and plants subjected to salt stress with the application of the cake product, both conditioned and unconditioned (Table 4.5.3). In contrast, the highest foliar MDA values were found in plants subjected to salt stress without cake application (Table 4.5.2). Like MDA, the highest foliar concentrations of H2O2 and O2- were observed in plants subjected exclusively to salinity (Table 4.5.2), while the application of the conditioned and unconditioned cake product resulted in a significant reduction in the foliar concentrations of these ROS in both cases under salinity conditions (Table 4.5.2). Finally, in general, the oxidative stress data confirm the positive and protective effect of the cake product applied both conditioned and unconditioned under salinity stress conditions, explaining the increased growth in those plants treated with this product under stress conditions.

Table 4.5.2: Indicators of oxidative stress

To prevent damage, plants employ ROS detoxification mechanisms, which can be divided into enzymatic systems and non-enzymatic systems composed of antioxidant compounds such as ascorbic acid, glutathione, phenols, flavonoids, anthocyanins, etc. The degree to which antioxidant enzyme activity and the amount of antioxidants increase under abiotic stress conditions such as water and salt stress can vary greatly between plant species and even between cultivars of the same species. The level of response and its contribution to resistance to water and salt stress therefore depend on the species, the development and metabolic status of the plant, as well as the duration and intensity of the stress. In this example, the response of the compounds phenols, ascorbate and glutathione in their total form, as well as the antioxidant capacity of plants, is analyzed by determining the FRAP and TEAC tests, in the improvement of resistance to saline stress, and whether the application of the biostimulant cake can influence the induction of plant resistance to this abiotic stress.

In general, Table 4.5.3 shows that the presence of salinity significantly induces an increase in the antioxidant compounds analyzed: phenols, ascorbate, and glutathione, in all treatments (with and without cake), with the lowest concentrations found in control plants not exposed to salinity (Table 4.5.3). These a priori data suggest that the increase in these antioxidant compounds is indeed due to an attempt to prevent the oxidative damage caused by adverse growth conditions in pepper plants. It is worth noting that the highest values for all the antioxidant compounds analyzed were found in the salinity treatment combined with the application of the conditioned cake (Table 4.5.3). On the contrary, the application of unconditioned cake under salinity conditions did not produce such biostimulation, since, although the concentration of phenols, ascorbate and glutathione increased with respect to the non-stressed control plants, it did not result in higher values with respect to those obtained in plants subjected exclusively to salinity (Table 4.5.3).

Table 4.5.3: Antioxidant compounds

The results for these antioxidant compounds coincide when analyzing antioxidant capacity using the FRAP and TEAC tests, which generally indicate the antioxidant activity of plants. As can be seen in Table 4.5.3.1, the highest values for these tests were found in plants subjected to salinity (NaCl treatment) and salinity with the conditioned application of the cake product (NaCl Cake_A treatment), especially for the FRAP test (Table 4.5.3.1). This is directly related to the highest concentrations of the antioxidant compounds phenols, ascorbate, and glutathione observed in this treatment (Table 4.5.3). As for the antioxidant compounds (Table 4.5.3), the effect of the unconditioned application of the cake product under salinity conditions inducing the antioxidant capacity was not observed when applying the unconditioned cake product in the lower FRAP and TEAC antioxidant test values (Table 4.5.3.1).

Table 4.5.3.1: Antioxidant capacity

In addition to antioxidant activity, the analysis of compounds such as proline are often good indicators of resistance to salt stress, as it often plays an osmoprotective, osmoregulatory, and antioxidant role against ROS generation. Proline levels in pepper plants largely reflected the degree of plant stress, with the highest values observed in plants subjected to salt stress without the application of the meal product (Table 4.5.3.2), and the lowest values observed in non-stressed control plants and in plants subjected to salt stress along with the application of the primarily conditioned meal product (Table 4.5.3.2), which were the treatments that showed the highest biomass production (Table 4.5.1). These results therefore suggest that in this assay, proline acted more as an indicator of the degree of plant stress than as an inducer of resistance to salt stress conditions. However, various studies have shown that in abiotic stress situations, proline reduction could contribute to improving plant resistance to different stresses. Proline degradation by the enzyme proline dehydrogenase results in O2 consumption, reducing the likelihood of ROS generation, which is precisely what could be occurring in salt stress treatments with the application of cake with the lowest ROS levels (Table 4.5.2).

Table 4.5.3.2: Foliar concentration of Proline

In short, considering the stress indicators and antioxidant compounds thus far, we can conclude that in pepper plants, we clearly observe a very significant beneficial and biostimulating effect of the application of the cake product under salt stress conditions. The application of the cake product produces an increase in plant biomass under salt stress conditions, which is due to a decrease in foliar ROS concentrations, thereby reducing lipid peroxidation. In this sense, the application of the conditioned cake compound is highlighted as more effective because it more significantly induces the antioxidant capacity of the plants, which may explain the benefit and positive effect that this treatment produces in pepper plants subjected to salinity.

Generally, under environmental stress, a significant inhibition of photosynthesis usually occurs, and it has been shown in some plant species that the application of biostimulants reverses this inhibition and, therefore, restores normal plant growth.

In order to verify the possible positive effect of the cake product under salt stress conditions on the photosynthetic process in plants, in this project we studied different parameters that directly define photosynthetic activity such as photochemical activity through the fluorescence of Chlorophyll a (Chl a), and photosynthetic efficiency (using IRGA-LiCOR 6400).

Chl a fluorescence has been shown to reflect the photosynthetic status of the plant and the photosynthetic changes produced under the effects of stress. When metabolic alterations occur, the plant produces fluorescence to dissipate excess energy and prevent damage from the stress. One of the parameters derived from the analysis of Chl a fluorescence is the quantum yield of primary photosynthesis (QV/Fm), which is a good indicator of plant photosynthetic performance. In healthy plants not subjected to intense stress, the QV/Fm value is usually around 0.85. Analysis of Chl a fluorescence also provides us with several indices that define plant vitality. Thus, high values of the QC/ABS ratio indicate a higher proportion of active reaction centers, making this an essential parameter in the functioning of the electron transport chain in photosystems. Continuing with indices related to plant vitality, we study the Plabs index, which is an index of photosynthetic functioning and represents the functionality of the two photosystems, and the 1-Vj value, which indicates the output of electrons primarily from photosystem II.

In table 4.5.4 we observe that in fact the control plants not stressed by excess salinity showed Fv/Fm values similar to 0.85, while the plants subjected to saline stress without application of the cake product showed the lowest values (Table 4.5.4), indicating a greater fluorescence of the chl a and therefore a greater degree of stress. Regarding the effect of the application of the cake product under salinity conditions, we observed how the plants showed values higher than 0.8, which suggests a better adaptation to the unfavorable growth conditions (Table 4.5.4).

The remaining indices that indicate plant photochemical activity and vitality through the functioning of the photochemical phase of photosynthesis suggest that under saline stress conditions, the application of both conditioned and unconditioned cake results in better coupling of the different components of the photochemical stage and improved efficiency in the transformation of light energy into chemical energy, thereby increasing plant vitality. Thus, the highest values for the RC/ABS and PIabs indices were found in control plants and in plants subjected to saline stress to which the specially conditioned cake was applied (Table 4.5.4). These results also indicate that electron loss in the photochemical phase of photosynthesis is reduced in plants subjected to saline stress to which the cake product was applied, implying a lower probability of ROS formation. However, these conclusions are not confirmed by the 1-Vj data, which is an index that primarily indicates the loss of electrons from photosystem II. As we can see, the 1-Vj values were similar across all treatments, with no significant differences (Table 4.5.4). These results are logical since this index is primarily influenced by environmental stresses, such as light radiation and heat stress.

Table 4.5.4: Chl a fluorescence parameters

Continuing with photosynthesis, measurements using a LICOR 6800 Portable Photosynthesis System infrared gas analyzer show the values of parameters that determine photosynthetic efficiency, such as transpiration rate (E), stomatal resistance (r), water use efficiency (WUE), and net photosynthetic rate (A). These parameters are crucial for plant adaptation to any type of stress.

When plants begin to experience salinity stress, leaf water loss decreases through a significant reduction in transpiration rate, increasing stomatal resistance due to stomatal closure. Stomatal closure is considered a rapid adaptation mechanism to water stress and is essential for reducing plant water loss. However, long-term maintenance of this strategy is generally counterproductive, as stomatal closure reduces the entry of intracellular CO2, leading to a reduction in photosynthesis (especially the Calvin cycle) and, consequently, a lack of the endogenous electron acceptor NADP, which ultimately leads to the formation of ROS.

Various studies indicate that the application of certain biostimulants under saline stress conditions could reverse this situation, since they prevent the complete closure of stomata under stress conditions. This would promote the maintenance of photosynthetic activity in plants, thereby reducing the massive generation of ROS under these conditions. The data obtained in this experiment confirm that the application of the specially conditioned cake product would act in this way, since its use under saline stress conditions increases the net photosynthesis rate (A), the transpiration rate (E), and water use efficiency (WUE), also reducing stomatal resistance (r) compared to plants treated only with saline stress (Table 4.5.5). This effect can also be observed, although less clearly and significantly, in stressed plants treated with the unconditioned cake product (Table 4.5.5). As expected, salinity-stressed treatments, regardless of the application of the cake product, showed greater water use efficiency (WUE) compared to non-stressed control plants (Table 4.5.5), mainly due to a reduction in stomatal opening and lower transpiration (Table 4.5.5).

Table 4.5.5: Photosynthetic efficiency parameters

In short, and from a photosynthetic efficiency perspective, under salt stress conditions, the application of the cake product, especially when it has been conditioned, would be very beneficial as it would produce:

(i) increased protection and activation of the photochemical process under stress conditions,

(ii) a greater availability of intracellular CO2 by reducing stomatal closure which would lead to adequate maintenance of photosynthesis (especially the Calvin Cycle), and therefore to a greater availability of the endogenous electron acceptor NADP which would reduce the transfer of electrons to oxygen, and therefore the formation of ROS,

(iii) In addition, the greater availability of intracellular CO2 in stressed plants treated with the cake product, especially when it has been conditioned, would produce a higher net photosynthesis rate, which would contribute under these stress conditions to the increase in plant biomass production (Table 4.5.1).

In contrast, plants subjected exclusively to salt stress produced a significant reduction in transpiration and an increase in stomatal resistance (Table 4.5.5), clearly indicating that this type of stress produces significant stomatal closure in order to prevent massive water loss. The long-term action of these processes in these plants causes a significant reduction in the net photosynthesis rate (Table 4.5.5), which leads to increased ROS formation (Table 4.5.2) and a significant reduction in biomass production in these stressed plants (Table 4.5.1).

Finally, in the case of salinity, the negative impacts of this abiotic stress on plant growth and development are due to physiological and metabolic alterations that are generally caused by both osmotic stress and ionic toxicity. The ionic toxicity of NaCl is caused by an ionic imbalance in the plant, due to an increased accumulation of sodium (Na+) and chloride ions (Cl-), to toxic levels, which causes a reduction in the absorption of other essential ions for plants such as potassium (K+). The high concentrations of Na+ and Cl- generated by saline stress are toxic to cellular metabolism and can inhibit the activity of many essential enzymes included in photosynthesis, cell division and expansion, disorganize the membrane structure, and ultimately this can lead to the inhibition of plant growth. These high concentrations of Na+ and Cl- also lead to the formation of ROS, such as H2O2 and O2-, drastically halting metabolic homeostasis and cell membrane integrity.

Several publications confirm that the use of biostimulants could be an effective tool for reducing the toxic effect of salinity on plants, due in part to a reduction in the absorption and accumulation of Na+ and Cl- ions. Table 4.5.6 shows that the application of conditioned cake in particular produces a very significant reduction in foliar Na+ and Cl- concentrations compared to plants treated with salinity, which showed the highest values (Table 4.5.6). It is also worth noting that the application of conditioned cake induced an increase in foliar K+ concentration under salinity conditions (Table 4.5.6). Therefore, the application of conditioned cake and its adsorptive effect could act at the root level, enhancing ionic selectivity processes by regulating the absorption and/or accumulation of Na+ and Cl- in the roots, which would reduce their translocation to the aerial parts, increasing that of K+. According to some studies, the additional contribution of organic compounds such as amino acids and/or hormones, such as cytokinins, derived from the application of biostimulants could explain the ionic selectivity processes that occur in roots, which lead to a reduction in the ionic toxicity of NaCl in the aerial parts of plants. The reduction in the foliar concentration of Na+ and Cl- ions and the increase in the foliar concentration of K+ (when conditioned cake is applied) (Table 4.5.6), together with the activation of other resistance processes explained throughout this report, would explain, in the case of saline stress, the improvement in the growth of plants to which the cake product is applied.

Table 4.5.6: Foliar concentration of Na+, Cl- and K+ ions

In this example, where a pepper crop was subjected to salt stress consisting of 100 mM NaCl, we clearly observed a very significant beneficial and biostimulating effect from the application of the cake product, especially when it was conditioned.

Under salt stress conditions, the application of cake significantly increased plant growth, increasing shoot biomass production and leaf area, which were higher than those obtained by stressed plants without cake application. Therefore, the beneficial effect of cake, especially when conditioned, in reducing the phytotoxicity of salt stress is primarily due to the following physiological mechanisms of action:

(i) Induction of antioxidant capacity and synthesis of antioxidant compounds which, together with the reduction of ROS formation by an increase in photochemical and photosynthetic efficiency, and by a decrease in foliar Na+ and Cl- concentration, would prevent oxidative damage and growth reduction,

(ii) Maintenance of photochemical activity and stimulation of photosynthetic efficiency which, together with a higher foliar concentration of K+ and a lower stomatal closure under conditions of saline stress, would allow maintaining a high net photosynthesis rate and thus reduce the generation of ROS, as well as counteracting the phytotoxic effect of Na+ ions.

In conclusion, we can indicate that the application of cake, whether conditioned or unconditioned, will always have a positive effect on salinity stress in pepper plants due to the large number of biostimulant compounds that have activity in the plant's physiology and metabolism. However, the results of this study indicate that conditioning the cake increases the availability and synergies of the biostimulant compounds, and significantly better results are observed than with the unconditioned cake. This may be due both to the improved agronomic applicability of the conditioned cake, as it does not generate foam and dissolves perfectly due to its absence of lumps/caking, and to the compound adsorption, slow release, and soil aeration effects produced by perlite.

4.6 Results and discussion of the experiment on limitation of nitrogen fertilization

The parameters that most reliably define the N nutritional status of plants are those related to plant growth. In this example, and to test the effect of CSL-B and Cake_A under different N fertilization doses, we analyzed the following parameters: biomass production or growth, expressed as fresh and/or dry biomass of the shoot, and leaf area. All these parameters reliably indicate plant growth under different growing conditions.

As we can see in table 4.6.1, which reflects the biomass production data of the aerial part and the leaf area, we observe that under 100% N fertilization (8 mM) the root application of the CSL-B and Torta_A products enhance plant growth, although the differences between the treatments were not significant from a statistical point of view.

Comparing the control treatments, the use of limiting levels of N (Control N-50% Table 2, Control N-25% Table 3) with respect to the optimal levels of fertilization (Control N-100% Table 1) produced, as expected, a significant reduction in the production of biomass of the aerial part as well as the leaf area (Tables 4.6.2 and 4.6.3), due to the fundamental role that N fertilization has in plant growth. As occurred under 100% N fertilization, root application of CSL-B and Torta_A under 50% and 75% N fertilization reductions resulted in increased plant growth compared to the N-50% control (Table 4.6.2) and N-25% control (Table 4.6.3) treatments, with root application of Torta_A being the most significant enhancer of both shoot biomass production and leaf area (Tables 4.6.2 and 4.6.3). Finally, it should be noted that under no circumstances under N-deficient conditions (N-50% and N-25%) did the application of the treatments restore plant growth compared to the N-100% control plants (Tables 4.6.2 and 4.6.3).

Table 4.6.1: Biomass production of the aerial part and leaf area in pepper plants subjected to different treatments with N-100% (8 mM N)

Table 4.6.2: Shoot biomass production and leaf area in pepper plants subjected to different treatments with 50% N (4 mM N)

Table 4.6.3: Shoot biomass production and leaf area in pepper plants subjected to different treatments with 25% N (2 mM N)

In short, considering the biomass and leaf area data obtained in this experiment 3, we can conclude:

(iii) The analyzed products CSL-B and Torta_A, applied to the roots, present biostimulant effects, although not statistically significant, on the growth of pepper plants under an optimal dose of N (N 100%) as fertilization, and in no case did phytotoxic effects occur.

(iv) Especially the product Torta_A applied via the root under N limiting conditions (N-50% and N-25%) significantly improves the growth of pepper plants, which clearly indicates a significant beneficial effect of this product defining it as a biostimulant for plants. This biostimulant effect can also be observed with the application of the product CSL-B via the root, although to a lesser extent. (v) Despite the biostimulant effect of the application of the product Torta_A under N deficient conditions (N-50% and N-25%), the application of this compound does not suppose a restoration of plant growth with respect to the control plants N-100%, so it should not be used to replace nitrogen fertilization.

NO3 is the predominant source of N for plants in most agricultural soils. It is absorbed by the roots and transported to the leaves, where it is transformed into assimilation products such as amino acids and proteins, necessary for biomass production. The first step in the assimilation of NO3- is its reduction to NH4+ in two reactions: first, NO3- is converted into NO2- by nitrate reductase (NR) (EC.1.6.6.) and subsequently nitrite reductase converts it to NH4+, being necessary reducing power in the form of NADH and reduced ferredoxin for the reaction. The NH4+ produced is assimilated in organic form by two enzymes: glutamine synthetase (GS) (E.C.6.3.1.2.) and glutamate synthase (GOGAT) (E.C.1.4.1.13) that produce glutamine (Gln) and glutamate (Glu) respectively, and which will be the precursors for the synthesis of other amino acids, nucleic acids, polyamines, chlorophylls, and hormones. In the GS/GOGAT cycle, GS incorporates NH4+ onto Glu, producing a Gln molecule with the expenditure of ATP. GOGAT then, with the expenditure of reducing power, catalyzes the transfer of the amide group from Gln to carbon 2 of α-ketoglutarate, producing two Glu molecules, one of which will restart the cycle.

The result of NH4+ incorporation can be quantified by analyzing organic N, which is generally the product of N assimilation and consists primarily of amino acids and proteins. Therefore, these parameters are also essential and effective for determining plant nutritional status.

Some research has shown that the application of biostimulants based on the presence of amino acids can improve plant growth and productivity by stimulating the assimilation processes of certain essential nutrients such as N, through the induction of enzymes such as NR and GS, both main regulators of this physiological process, and the synthesis of organic nitrogen compounds such as amino acids and soluble proteins.

In this study we can see that under adequate N fertilization conditions (N-100%), the use of the treatments produced few variations in the parameters related to N assimilation (Table 4.6.4). Specifically, NR activity did not vary with any treatment and only with the application of the product Torta_A via the root was there a significant increase in GS activity (Table 4.6.4). Finally, and under these adequate N fertilization conditions (N-100%), the use of the products CSL-B and Torta_A, via the root, did produce significant increases in foliar protein concentrations, with no changes in amino acid values compared to control plants (Table 4.6.4). The increased concentration of soluble proteins observed in these treatments (CSL-B and Torta_A) could be very beneficial for plant development, since these compounds include proteins with enzymatic activity such as RUBISCO, which are essential for the photosynthetic process and carbon metabolism. Ultimately, these results could explain the stimulation of plant growth observed under adequate N conditions when these treatments are applied (Table 4.6.1).

Table 4.6.4: N assimilation parameters in pepper plants subjected to different treatments with N-100% (8 mM N)

Under N-limiting conditions (N-50% and N-25%), we can first observe how all parameters defining N assimilation are significantly reduced compared to those obtained in control plants fertilized with adequate N levels (N-100% control) (Tables 4.6.5 and 4.6.6). In addition to this behavior, we observed that the general application of CSL-B and Torta_A products, via the root, significantly increased N assimilation in plants subjected to N deficiency (in both N-50% and N-25% treatments, Tables 4.6.5 and 4.6.6, respectively), increasing NR and GS activities, as well as foliar concentrations of amino acids and soluble proteins, compared to the values obtained in N-50% and N-25% control plants (Tables 4.6.5 and 4.6.6, respectively). It should be noted that under these conditions of N limitation (N-50% and N-25%) it was mainly the Torta_A product applied via the root system that resulted in a greater induction of the NR and GS enzymes that regulate the N assimilation process (Tables 4.6.5 and 4.6.6).

Table 4.6.5: N assimilation parameters in pepper plants subjected to different treatments with N-50% (4 mM N)

Table 4.6.6: N assimilation parameters in pepper plants subjected to different treatments with 25% N (2 mM N)

Both amino acids and soluble proteins are essential for their effect on plant growth, performing functions in primary metabolism such as: chlorophyll synthesis, increasing photosynthetic activity, maintaining water relations in the plant, synthesis of growth hormones such as auxin, precursors of other amino acids, and protein synthesis. In addition to these primary metabolism functions, these nitrogen compounds also intervene in the induction of secondary metabolism generating defense compounds such as phenols, alkaloids, etc. Therefore, the increase we observed with the application of the products CSL-B and Torta_A, via the root, on the foliar concentration of amino acids and soluble proteins under N limiting conditions (N-50% and N-25%) (Tables 4.6.5 and 4.6.6), could explain the increase in biomass production of pepper plants treated with this biostimulant under N-deficient conditions in the medium (Tables 4.6.2 and 4.6.3).

Knowing the NO3- status in leaves is an essential indicator of N assimilation processes, especially when nitrogen fertilization is carried out in the form of NO3- as is the case in this trial. In general, and under normal N fertilization conditions, the reduction in NO3- concentration in leaves is usually indicative of an increase in N assimilation processes, since the decrease in foliar NO3- concentration is directly related to an induction of the main enzymes that carry out this physiological process, such as NR and GS. Indeed, under optimal N conditions (N-100%), it was the Torta_A treatment applied via the root that showed the lowest levels of foliar NO3- concentration (Table 4.6.7), which could be perfectly explained by the increase in N assimilation with greater GS activity in this treatment (Table 4.6.4). On the contrary, and with respect to the concentrations of organic N and total N, all treatments presented similar foliar concentrations with no differences observed (Table 4.6.7), which indicates that the application of these products does not modify the nutritional status of these forms of N in the plants.

Table 4.6.7: Foliar concentration of the different forms of N in pepper plants subjected to the different treatments with N-100% (8 mM N)

Regarding the NO3 concentration in plants subjected to N-limiting conditions (N-50% and N-25%), we observed that the NO3 concentration was similar in all N-50% treatments (Table 4.6.8), while in plants with N-25%, the application of the Torta_A product applied via the roots increased the NO3 concentration (Table 4.6.9). In all cases, as expected, the foliar NO3 concentration was lower than that obtained in control plants with N-100% (Tables 4.6.8 and 4.6.9). Regarding organic and total N, only the application of the Torta_A product resulted in an increase in the organic and total N concentration compared to plants treated with N-50% and N-25% (Tables 4.6.8 and 4.6.9). As occurred for the concentration of NO3, all plants treated with N-50% and N-25% presented lower concentrations of organic and total N than plants treated with N-100% (Tables 4.6.8 and 4.6.9).

Table 4.6.8: Foliar concentration of the different forms of N in pepper plants subjected to the different treatments with N-50% (4 mM N)

Table 4.6.9: Foliar concentrations of different forms of N in pepper plants subjected to different treatments with 25% N (2 mM N)

In short, these results indicate that root application of Torta_A under N-deficient conditions (N-50% and N-25%), in addition to inducing greater synthesis of amino acids and soluble proteins (Tables 4.6.5 and 4.6.6), leads to improved N nutritional status by stimulating increased foliar concentrations of total N and organic N (Tables 4.6.8 and 4.6.9). Although further research is needed, these results may possibly be explained by a possible stimulation of N uptake by root-applied Torta_A under N-limiting conditions in the growing medium (N-50% and N-25%).

Currently, the importance of nitrogen is magnified because current agriculture is dominated by production, as recent decades have seen a rapid population explosion, generating a growing need for more plant products for human consumption. All this must be done without expanding the amount of cultivated land, as agriculture currently occupies most of the Earth's fertile land and uses a large portion of the resources, such as water and fertilizers, necessary for its maintenance. This situation has generated a dynamic in which excessive fertilizer application has become a common agronomic practice, especially in the case of nitrogen fertilizers, due to the fundamental role this element plays in crop production. For example, in 2018, global demand for synthetic nitrogen fertilizers exceeded 200 million tons (FAO: https://www.fao.org/news/story/es/item/277654/icode/).

Furthermore, the inefficient use of nitrogen fertilizers has gradually increased proportionally to demand in recent decades, generating serious environmental pollution. The most common impact produced by the misuse of nitrogen fertilizers is the leaching of NO3- into underground aquifers, which causes eutrophication of freshwater and marine ecosystems. Furthermore, gaseous nitrogen oxides are generated that can reach the troposphere and react with ozone, producing toxic ammonia emissions. Furthermore, NO3- also poses a risk to human health since, when ingested, it is rapidly transformed into nitrite and nitroso compounds. These forms are toxic and can cause serious pathologies such as methemoglobinemia or blue baby syndrome, or an increased risk of cancer when nitrites are transformed into nitrosamines in the human body.

These risks are accentuated if we take into account that plants are only able to convert 30-40% of the applied N into a product intended for human consumption, subsequently producing an accumulation of NO3- mainly in the leaves, affecting the nutritional quality of the so-called "leafy vegetables", such as lettuce, spinach... Furthermore, another problem with nitrogen fertilizers is that their efficiency in agriculture is generally relatively low, with the efficiency in the use of nitrogen fertilizers in agriculture being, in the best of cases, between 70%-80%.

Therefore, considering all the problems associated with nitrogen fertilizers, it is essential to: (i) properly manage N fertilization in relation to the use of the dose, application time and choice of the appropriate chemical form, and (ii) use agronomic techniques that lead to an increase and improvement of nitrogen use efficiency (NUE) by plants. NUE is defined as the biomass production per unit of available N. This can be divided into two fundamental processes: (i) the ability of the plant to absorb N from the soil (N uptake efficiency - NUpE) and (ii) the efficient use of the absorbed N, which is defined as the plant's ability to transfer and use this element in the production of biomass of the different plant organs (N utilization efficiency - NUtE).

Considering all the above, higher NUE could improve crop yield and quality, while reducing economic costs and decreasing environmental degradation caused by the application of N fertilizers. In recent years, the focus for improving NUE in crops has been on biotechnology and plant breeding strategies, but it is also currently necessary to evaluate alternative, rapid, effective and environmentally friendly means such as the use of biostimulants.

When N fertilization was optimal (N-100%), the application of the products CSL-B and Torta_A, via the roots, did not lead to an improvement in the different parameters that determine the NUE (Table 4.6.10), which would possibly explain the lack of significant growth stimulation that we observed in the plants treated with the product (Table 4.6.1).

Table 4.6.10: NUE and RAN in pepper plants subjected to different treatments with N-100% (8 mM N)

Under N-limiting conditions in the growing medium (50% N and 25% N), the application of the treatments significantly improved the various parameters that define NUE (Tables 4.6.11 and 4.6.12). Thus, application of the CSL-B product most significantly stimulated N uptake efficiency (NUpE), especially under 50% N-deficient conditions (Table 4.6.11). In contrast, application of the Torta_A product applied via the roots induced a significant increase in N utilization efficiency (NUtE) and NUE under 50% and 25% N-deficient conditions (Tables 4.6.11 and 4.6.12). It should be noted that both the application of the CSL-B product and Torta_A improves all NUE parameters under N-limiting conditions in the culture medium (Tables 4.6.11 and 4.6.12), which would explain the biostimulant effect of this compound inducing an increase in the biomass production of the aerial part under N-50% and N-25% limiting conditions, especially when Torta_A is applied (Tables 4.6.2 and 4.6.3).

Table 4.6.11: NUE and RAN in pepper plants subjected to different treatments with N-50% (4 mM N)

Table 4.6.12: NUE and RAN in pepper plants subjected to different treatments with 25% N (2 mM N)

In short, and considering all the studied parameters related to NUE (Tables 4.6.11 and 4.6.12), we can define that the application of the CSL-B and Torta_A products would constitute a very useful technique in the development of crops in N-limiting areas, enhancing NUE through: (i) an improvement in the efficiency of N use

N in plants (NUtE) in the case of Cake_A, and (ii) by improving the absorption of available N (NUpE) in the case of CSL-B.

The results obtained in this experiment show significant scientific evidence that supports the following conclusions:

(i) Under appropriate conditions in the fertilization with NO3" for a pepper crop (N-100%), the use of the products CSL-B and Torta_A shows a limited beneficial effect on the plants, increasing, although not significantly, the biomass production of the aerial part and not presenting any effect on the NUE.

(ii) Under N limiting conditions (N-50% and N-25%) especially the use of the Torta_A product would be the most suitable and beneficial for a pepper crop, since it substantially improves plant growth under these conditions, being the mechanisms of action: the induction of N assimilation (enzymatic activities NR and GS) and increase in the synthesis and accumulation of amino acids and proteins, significant improvement in N use efficiency (NUtE and NUE especially), and increase in the apparent recovery of available fertilized N (RAN). Therefore, the use of the Torta_A product would constitute a very useful technique in the development of crops in N limiting areas.

(iii) Finally, under N limiting conditions (N-50% and N-25%) the application of the product CSL-B is also beneficial in other aspects, but to a lesser extent than the effect presented by the product Torta_A. The application of CSL-B would be very useful in improving the NUE through the stimulation of the absorption processes of available N in the culture medium (NUpE and RAN).

Claims (20)

1. Process for treating or adapting a corn steep liquor filter cake to produce a powder cake, said process comprising the following steps: a) stir a quantity of corn steep liquor filter cake (at a speed of between 10 rpm and 1000 rpm) and, maintaining these conditions, add a preservative in a quantity of between 0.1% and 1% by weight with respect to the weight of the starting cake, b) stirring the mixture obtained in (a) at a speed between 10 rpm and 1000 rpm for a time between 1 min and 120 min; c) drying the mixture obtained in (b) until obtaining a product with a moisture content between 0.1% and 10% by weight, d) grinding the product obtained in (c) to obtain said product in a powdery state. 2. Method according to claim 1, wherein the stirring time of step (b) is at least 15 min. 3. Method according to claim 1 or 2, wherein the added preservative is selected from the following: citric acid, potassium sorbate, octylisothiazolinone (OIT), (ethylenedioxy)dimethanol (EDDM), iodopropynyl butylcarbamate, phenoxyethanol, benzoisothiazolinone, 2-(thiocyanomethylthio)-benzothiazole, orthophenylphenol, parachlorometacresol, methylchloroisothiazolinone, methylisothiazolinone, glutaraldehyde and any combination of the above. 4. Method according to any of the preceding claims, wherein the added preservative is octylisothiazolinone, (ethylenedioxy)dimethanol or a combination thereof. 5. Method according to any of the preceding claims, wherein step a) and/or b) are carried out in a Cowles type shaker or in a high shear homogenizer. 6. Method according to any of the preceding claims, wherein the drying step c) is carried out in a vacuum paddle dryer. 7. Method according to any of the preceding claims 1 to 5, wherein the drying step c) is carried out by adding a hygroscopic compound to the mixture obtained in step b). 8. Method according to claim 7, wherein the hygroscopic compound is selected from the list comprising: Lemna sp., macroalgae of the genus Ascophyllum sp., Ecklonia sp., Macrocystis sp. or Laminaria sp., cellulose, polyacrylates, diatomaceous earth, perlite, vermiculite, maltodextrins, silica and kaolin. 9. Method according to claim 7 or 8, wherein the hygroscopic compound is added in an amount of between 10% and 90% by weight with respect to the cake obtained in b). 10. Method according to any of the preceding claims, wherein, after the drying of step c), an antifoam is added. 11. Method according to claim 10, wherein the antifoam is a powder antifoam based on a combination of polyether-polyols and amorphous silica. 12. Method according to claim 10 or 11, wherein the antifoam is added in an amount between 0.1% and 2% by weight with respect to the dried cake obtained in c). 13. Method according to any of the preceding claims, wherein the starting cake from step a) is obtained by a pressed filter process of the corn maceration liquor comprising the following steps: - a solution of perlite between 0.1% and 5% by weight is made in water with constant stirring, - the previous solution is passed through a filter press to form a pre-layer of perlite on the filter itself, - on the other hand, between 0.1% and 5% by weight of perlite is added to the corn steep liquor, - the corn steep liquor is filtered under pressure between 10 and 15 bars, - the filter cake retained in the filter is recovered. 14. Method, according to claim 13, where, prior to adding perlite to the corn maceration liquor, the latter is kept under agitation in the air for between 1 and 30 days. 15. Corn steep liquor cake obtained according to the process described in any of claims 1 to 14. 16. Cake, according to claim 15, wherein said cake is in a powdery state and has a moisture content of less than 10% by weight and a preservative content of between 0.1 and 2% by weight. 17. Cake according to claim 15 or 16, comprising perlite in an amount between 0.5% and 5% by weight. 18. Cake according to any one of claims 15 to 17, comprising an antifoam in an amount of between 0.1% and 3% by weight. 19. Use of a cake described in any of claims 15 to 18 as a plant fertilizer or biostimulant. 20. Fertilizer characterized in that it comprises the corn steep liquor cake described in any of claims 15 to 18.