WO2015076688A1 - Process for obtainment of fertilizers from vinasse - Google Patents

Abstract

The invention refers to a process for obtainment from vinasse of an organo- mineral fertilizer that contains humic acids, and a nitrogen fertilizer with osmo- protectant action due to the presence of (glycine)betaine. The process consists of the following steps: separation of an yeast concentrate from vinasse; high pressure homogenization of yeast concentrate; addition of steel slag in clarified vinasse and maintaining vinasse on aerated and agitated medium, in order to promote the catalytic action of manganese and iron oxides present in steel slag on the reaction of humification of fulvic acids to humic acids; precipitation of humic acids by addition of bio-char and acidification; separation of the filtrate and use as osmo-protectant fertilizer; pelletizing of the precipitate together with yeast homogenate, bio-char and softwood sawdust, in order to obtain an organo-mineral fertilizer.

Description

PROCESS FOR OBTAINMENT OF FERTILIZERS FROM VINASSE

This invention refers to a process for obtainment of fertilizers from vinasse, (co)product from the utilization of molasses in industrial fermentative processes, especially from the production of (bio)ethanol from molasses, respectively of an organo-mineral fertilizer which contains humic acids, and a nitrogen fertilizer with osmo-protectant action, due to the presence (glycine)betaine.

There are known different processes intended to exploit as agro-fertilizers the compounds accumulated into vinasse, which have a high biological value for plants. Potassium, present in vinasse in quantities that can reach levels of over 1%, represents a major nutrient for plants. (Glycine)Betaine, present in molasses in large quantities, unused by yeasts during alcoholic fermentation, and concentrated into vinasse, is a donor of methyl groups, involved into the acceleration of the methionine cycle (methionine - S-Adenosyl-methionine - homocysteine - methionine). This cycle of methionine have a regulatory function on: (/) gene expression through methylation, including epigenetic response to environmental factors, and (//) the cascade of signal factors, at intra- and inter - cellular level, polyamines - nitric oxide - peroxynitrite. Due to this involvement in the regulation of the metabolism of some key compounds (glycine)betaine is a versatile compound, with a major function in plant response to abiotic and biotic stress (see e.g. the review Ahmad et al., 2013, Plant Biotech. Rep., 7: 49-57). The osmoprotectant effect, protecting plants against hydric stress due to low rainfall, drought and salinity, has significant implications in the current context of climate changes, characterized by modification of the pattern of rainfall and increased drought risk.

The technical problems related to the use of vinasse as fertilizers / fertigants are resulted from: (/) the microbiological instability of vinasse, due to the presence of fermentation yeast, partially inactivated by distillation, which makes vinasse difficult to be kept as it for more than 10 days; (/^'/) clastogenicity / chromosomal breakdown with aberrations, determined by the melanoidinic / humic-like (soluble) compounds, formed and accumulated during repeated heat treatments, included into technological processes that lead to the obtainment of molasses and then to vinasse (see e.g. da Silva et al., 2013, Environ. Monit. Assess, 185: 1627-1636); (//^'/) toxicity to aquatic organisms of soluble melanoidins from fertigants / vinasse applied as fertilizer, which can be leached from the soil into surface waters (e.g. Botelho et al., 2012, Tox. Environ. Chem. 94: 2035-2045); (/V) the negative modifications of soil characteristics, as a result of the application of vinasse, recently reviewed by Christofoletti et al., 2013, Waste Manag. 33: 3013-2761 , and especially the acidification of the soils and depletion in manganese; (v) significant risk of formation of additional toxic melanoidins, if microbiological stabilization is done through concentration by water evaporation, and the risk of pipes encrustation on heat exchangers and/or on evaporators, due to the precipitation of calcium and magnesium salts present in the vinasse; (w) the large amount of bacterial degradable organic carbon from some compositions of vinasse, which make the nitrogen from vinasse unavailable to plants; (vii) the need to ensure a high rate of scale-up processes, required by the significant vinasse amount formed during the production of (bio)ethanol from molasses - i.e. approx. 9 hi vinasse for 1 hi (bio)ethanol.

Patent US 5 851 405 describes a process of clarification and of recovery of betaine and potassium from vinasse. Clarification of vinasse is performed after pH adjustment, preferably from 6.5 to 7.5, on a centrifugal separator, at 9,000 x g. Clarified vinasse is concentrated in a circular evaporator, till a solid content of 59% to 65%. Potassium sulfate crystals, which are beginning to be formed from the 40% dry matter content in concentrated vinasse, are removed by decantation and filtration on a press filter, with diatomaceous earth as a filtration additive. The resulting concentrated vinasse is diluted and subjected to ion exchange chromatography separation for recovery of the betaine. The process involves concentration operations, followed by dilution of concentrated vinasse and re-concentration of chromatographic eluat containing betaine. During the concentration process, done at high temperatures, the risk of the formation of new melanoidinic (soluble) compounds is high. In the process described by U.S. Patent 5 851 405 are not claimed steps wherein these potentially dangerous organic compounds are inactivated.

Patent Application WO2010/070622 describes a process of electro- flotation/oxidation, which reduce total organic carbon, oxygen consumption and total solids of vinasse, including the potentially dangerous melanoidinic compounds, which involves the following steps: introduction of vinasse into electrolytic cells equipped with galvanized steel electrodes, adjustment of vinasse pH to a value between 7 and 14, preferably between 7 and 1 1 , the application of a constant current, at a current density of between 20 and 40 mA/cm², hydrogen peroxide injection, continuous or sequential, in the vinasse, in the proximity of the electrodes, in a concentration that is between 10 and 67,000 ppm H₂0₂ per liter of vinasse, preferably between 1 ,000 and 67,000 ppm H₂0₂ per liter of vinasse, removal of treated vinasse from electrolytic cell and separation of flocculated insoluble solids by gravity, filtration or centrifugation.

A drawback of the process described above is the use of relatively large quantities of hydrogen peroxide, relatively expensive compounds, which require precautions in utilization. Also the described process does not include subsequent steps of using of vinasse purified by the process of electro-flotation/oxidation.

The authors have improved the process described in the WO2010/070622 in a new Patent Application WO2011/158195, wherein the flocculation is promoted by the addition of an co-adjuvant, the supernatant is re-introduced in flocculation step to further reduce total organic carbon, electrodes are from iron, steel, carbon, aluminum, or a combination of these, the pH is on the acid range, between 4 and 5, the columbic charge of electrochemical reactor is between 5000 and 10000 coulombs per liter, at a current density between 0.3 and 2 mA cm², hydrogen peroxide is reduced to the max. 5000 ppm, and solids obtained by flocculation are used for (bio) fuels or as agro-fertilizers.

Improved process continues to use large quantities of hydrogen peroxide, max. 5000 ppm, is not exploiting for agricultural purposes the betaine, retained in the supernatant and subjected to repeated oxidative degradation, and melanoidinic compounds are not differentiated, according to their beneficial effects on plant or potentially (geno)toxic effects.

A portion of the organic matter removed through the above described process of electro-flotation/oxidation is represented by melanoidinic compounds from the category of humic acids - Muhammad et al., 2008, Chemosphere, 71 : 1730-1737. Humic acids have a significant role in the formation of soil - Tarchitzky et al., 2000, Soil Sci. 165, 327-342, and promote plant growth and nutrition - Nardi et al., 2002, Soil Biol Biochem., 34: 1527-1536, including due to effects which are similar to phytohormones. In order to exploit these compounds with beneficial effect on plants Patent Application WO 2009/129589 presents a process for obtainment of a organo- mineral granulated fertilizer, consisting of the following steps: evaporative concentration of vinasse up to 65% dry matter, mixing of the concentrated vinasse with filter cake resulted from sugar syrup fabrication from sugar cane, preferably up to 10 ...20% residual moisture, adding ash from the burning of sugar cane bagasse and/or other residue of sugar cane, adding macro-nutrients, primary (N, P, K) and secondary (Ca, Mg, S), and micro-nutrients (Zn, Fe, Cu, CI, Bo, Mn, Mo), to obtain various organo-mineral agrochemical formulations, corresponding to different types of applications, required by plants cultivation.

The process described above is applicable only in the factories which produce (bio)ethanol from sugar cane, and cannot apply to factories which produce (bio) ethanol from molasses, sugar cane or sugar beet, in which there is not produced filter cake from sugar syrup filtration and much less sugar cane bagasse / ash. The process involves steps wherein the energy consumption is high, such as the evaporative concentration of vinasse, and does not include steps to avoid further formation of potentially toxic melanoidinic compounds.

In the process described in the patent application WO 2009/129589 the melanoidinic compounds are not differentiated according to their effect, beneficial in the case of humic acids, molecular complexes with low solubility, or (potentially) toxic for fulvic acid, melanoidins with higher solubility in water. According to the new perspective of humic acids, revealed by the new techniques of investigation, described by Sutton and Sposito, 2005, Environ. Sci. Technol., 39: 9009-9015, humic acids are complex macromolecular melanoidinic compounds, stabilized by hydrophobic interactions and hydrogen bonds, insoluble at acidic pH and dissolved in alkaline pH, due to destabilization resulted from hydrophobic interactions, and fulvic acids are melanoidinic compounds soluble in water, both on acid and alkaline pH, with a significant presence of polar residues of (5-methyl)-2-furfural, formed by thermal dehydration of carbohydrate molecules. Humic acids, with low solubility and more pronounced hydrophobic character, have a beneficial effect on soil fertility and structure, as well as the stimulation of plant nutrition and culture - see e.g. Schnitzer and Moreal, 2011 , Advan. Agron, 1 13: 141-195. In the generic category called fulvic acids, melanoidinic compounds with a higher water solubility, and which have a higher capacity of transportation through various permeability barriers from various organisms, are included also those soluble melanoidins, which are responsible for clastogenicity / chromosomal breakdown with aberrations, and for the toxicity to aquatic organisms of leachates from soils fertilized / fertigated with vinasse.

Vinasse has a high content of fulvic acids and a reduced content of humic acids - Tejada and Gonzalez, 2005, Europ. J. Agron. 23: 336-347. The role of manganese in catalyzing abiotic humification process, demonstrated by Jokic et al., 2001 , Geophys. Res. Lett., 28: 4019-3902, explains (partially) also the manganese depletion on soils treated with large amounts of vinasse with soluble fulvic acids / incompletely humified melanoidins, that decrease the content of bio-available manganese in soils, as a result of humification reactions till humic acids.

It is therefore necessary to develop a process by which to exploit the agronomic benefits of humic acids from vinasse, limiting the risks associated with soluble melanoidinic compounds / fulvic acids from vinasse. The object of this invention is to describe such a process, wherein humification reactions of fulvic acids from vinasse to humic acids are performed, allowing a full recovery of melanoidins from vinasse, by obtaining organic / organo-mineral fertilizers, with reduced risks to the various components of the environment.

The process according to the invention consists of the following steps:

The separation of an yeast concentrate from vinasse, which contains at least 10% yeast, by using a centrifugal separator, operated at min. 8500 g, high pressure homogenization of yeast concentrate, homogenate refrigeration and maintenance of yeast homogenate to 4°C until further use;

Adjustment of the clarified vinasse pH to value 4, addition of steel slag, in a ratio of 2 parts steel slag to 98 parts vinasse, and maintainance at 60°C, with 20 rpm agitation and aeration with 0.2 liter air / liter medium/ min for 12 hours, in order to promote the catalyzation of the humification reaction, by the manganese and iron oxides present in steel slag;

Cooling of the mixture till 25°C, adjustment of pH in the mixture to 3.5, and addition of bio-char, in the ratio of 2 parts bio-char to 100 parts of vinasse - steel slag, and maintaining, with 20 rpm agitation for 3 hours;

Adjustment of the pH of the mixture vinasse - steel slag - bio-char to 1 .5 units, and maintaining at 25°C with 20 rpm agitation for 20 min, to complete the humic acids precipitation, pre-existing and formed by the catalytic action of iron and manganese oxides;

Filtration of the mixture of vinasse - steel slag - bio-char, on a rotative vacuum filter, with differential pressure of 0,6 MPa, with the separation of a filtrate (F), with high content of (glycine)betaine, usefull a nitrogen fertilizer F1 , and a precipitate (P); V Blending of the precipitate (P) retained on filter, with a residual moisture content of 20%, with bio-char, with a residual moisture content of 6%, with softwood sawdust, with residual moisture content of 14%, and homogenized yeast, with humidity of 85%, in the ratio of 6 parts precipitate (P) : 44 parts bio-char : 40 parts of softwood sawdust: 10 part homogenized yeast, and densification of the resulted mixture by compressing into a pellet press, with horizontal dyes, to form pellets with a length of approx. 15 mm and a diameter of 5 ... 8 mm, representing the organo- mineral agro-fertilizer F2.

The favorite aspects of the process described above are:

High pressure homogenization of yeast concentrate by passing through a high pressure piston homogenizer piston fitted with a knife edge valve, 2 cycles at 150 MPa and acidification to pH 2.0 of homogenate with phosphoric acid;

Steel slag used to catalyze the oxidation of fulvic acids and their coagulation as complexes of humic acids, contains at least 2.5% of manganese oxide and 25% iron oxides;

Bio-char used, with a specific microporous volume, W_a, of at least 0,6 cm³g^"\ and with a surface of micropores, S_mi_Cro, of min. 450 m²g^"1, is obtained by micro-wave assisted pyrolysis, from a mixture of distiller grains, from bioethanol production, and corn stover, 25 ...50% distiller grain: 50 ... 75% corn stover, and micro-wave assisted pyrolysis is performed by exposing the above mixture for 20 minutes to micro-waves, with a power of 1000 W and an incident frequency of 2450 MHz.

v Densification of the mixture, precipitate (P) : bio-char : softwood sawdust : yeast homogenate, is performed with a specific power of 1 kW for 0.015 ...0.02 m² surface of the matrix, maintaining the temperature of the pelletizing mixture of about 65°C.

The process, according to the invention, presents the following advantages:

Recover all the compounds from vinasse, which have high biological value for plants, including fermentation yeast, into agro-organic fertilizers;

Ensures a high stability during storage of agro-fertilizers, in the case of nitrogen fertilizer F1 due to the stabilization by low pH and the removal of easily degradable organic substances, and in the case of organo-mineral fertilizer F2 due to pelletizing in granules, with humidity of max. 15%.

Reduce the environmental risks associated with soluble melanoidins as a result of: (/) humification / oxidation, followed by association into humic acids complexes, catalyzed by manganese and iron oxides from steel slag and (//) fixing into the spongious structure of biochar of eventually remaining soluble melanoidins / incompletely humified under the catalytic action of steel slags; Avoids the processes of vinasse stabilization by heat treatment, i.e. evaporative concentration, which is energy intensive and potential generators of new soluble melanoidins;

In nitrogen fertilizer F1 reduce the ratio C:N, increasing the bioavailability of nitrogen for plants;

Does not involve very significant investment costs because it does not require dedicated equipment, but equipment used into various process industries, which, thanks to this larger market have a lower cost;

Permit an easy scale-up and high rate in real-time processing of large amounts of vinasse.

Below is an embodiment of the invention which illustrates it, without limiting it.

Example. From the vinasse collected from the distillation of the fermented molasses is separated an yeast concentrate, containing at least 10% yeast, by centrifugation on a continuous laboratory centrifuge, Westfalia Laboratory Separator, model SA 1 -02-175 (GEA Westfalia Separator Group, Germany, Oelde), which is operated at a speed of the disks of 10,000 rpm, equivalent of 8,500 x g; at a rate of 1 l/min, with continuous separation of clarified vinasse, and discontinuous of the yeast concentrate, when this reach a density of 1 100 kg/m³. The yeast concentrate is homogenized into a high pressure piston homogenizer GEA Niro Soavi NS2006 Arriete (GEA Niro Soavi, Parma, Italy) with a knife edge valve, two cycles 150 MPa. High pressure homogenization causes inactivation of microbial cells by disruption induced by pressure variations and passing through the knife edge valve. Yeast homogenate is cooled, acidified at pH 2.0 with phosphoric acid and maintained in a container chilled at 4°C until further use.

In a reaction vessel of 150 liters total capacity and 100 liters working capacity, with stirring, are added 98 kg of clarified vinasse, whose pH is corrected to 4, by the addition of phosphoric acid and potassium hydroxide. Vinasse is heated to a temperature of 60°C, and 2 kg of steel slag are added. The mixture is maintained at 60°C, with 20 rpm agitation, and aeration, 0.2 liters air / liter medium / min for 12 hours, in order to promote the catalyzation of the humification reaction, by the manganese and iron oxides present in steel slag.

Steel slag used contains at least 2.5% of manganese oxide and 25% iron oxides. The content of oxides of manganese and iron oxides are determined by using an X-ray wavelength dispersive fluorescence spectrometer, e.g. PW 4025 iniPal (PANalytical, Almelo, Netherland).

The mixture clarified vinasse - steel slag is cooled to 25°C, the pH is adjusted to 3.5 and are added 2 kg of biochar. It is maintained, under agitation at 20 rpm, for 3 hours.

Biochar used in the above mixture has a specific microporous volume, W_a, of at least 0.6 cm³g^~\ and a specific surface of micropores, Smicro. of min. 450 m²g^"\ and is obtained by micro-wave assisted pyrolysis of a mixture of distiller grains, from bioethanol production, and corn stover.

Microporous specific volume, W_a, of biochar was calculated from the linear function Dubinin-Radushkevich (Stoeckli et al., 2001 , Carbon 39: 1 15-1 1 16), after determination of adsorption isotherm in liquid nitrogen at 77 K, using a physisorption analyzer (Micromeritics ASAP 2020, Micromeritics, Norcross, GA, USA). Specific surface of micropores was estimated by reporting the values obtained for the specific microporous volume to average dimensions of micropores (Stoeckli, 1996 in: Patrick, J. (ed.), Porosity in Carbons - Characterization and Applications. Arnold, London, pp. 67-92).

Any other type of bio-char with capacities of absorption of melanoidinic compounds, which has the characteristics mentioned above, can be used in this example.

The pH of the mixture vinasse - steel slag - biochar is adjusted to 1.5 pH units, is maintained at 25°C, under 20 rpm agitation, for 20 min, to complete the humic acids precipitation, pre-existing and formed by mediated catalysis of iron and manganese oxides. The mixture of vinasse- steel slag - biochar is separated on a pressure filter (RPF T01 , BHS, Sonthofen-Sonthofen, Germany), at 0.6 MPa, separating a filtrate F1 , which is further used as fertigant with high (glycine)betaine content, and precipitate (P). 6 kg of the precipitate (P) retained on the filter, with a residual moisture content of 20%, is mixed in a mixer (FG3 Unimec, Siena, Italy), with 44 kg biochar with residual moisture of 6%, with 40 kg of softwood sawdust, with residual moisture of 14%, 10 parts of homogenized yeast with humidity of 85%. The mixture is densified by using a pellet press with horizontal dyes, model Kahl 14-175 (Amandus Kahl, Reinbek/Hamburg, Germany), at a specific power of 1 kW for 0.015 ...0.02 m², maintaining the temperature of the mixture of pellets at approximately 65°C, to form pellets with a length of approx. 15 mm and a diameter of 5....8 mm, which represents the agro-fertilizer organo-mineral F2.

In the vinasse clarified by centrifugation and in F1 filtrate were determined several chemical characteristics. Organic matter was determined according to SR EN 13039: 2012. For the determination of humic acids and fulvic form vinasse and from filtrate F1 were collected samples which were extracted with 0.1 M sodium pyrophosphate and 0.1 sodium hydroxide. Supernatant was acidified to pH 2 with HCI and kept overnight at room temperature. In order to separate the humic acids and fulvic acids, the solution was centrifuged, and humic acids precipitate was dissolved with 2 M sodium hydroxide (Yeomans and Bremner, 1988, Comm. Soil Sci. Plant Anal., 19: 1467-1476). The content of carbon in humic acids and in fulvic acids was determined by method of Sims and Adonis, 1971 , Soil Sci., 1 12: 137-141. Total phosphorus was determined in accordance with EN 13346: 2002, total nitrogen was determined by Kjeldahl method and the potassium and sodium were determined by flame photometry. Betaine-glycine content was determined by high-pressure liquid chromatography, according to the method described by Chendrimada er a/., ^'2002, J. Sci. Food Agric, 82: 1556-1563. The results are presented in table 1 below.

Tab. 1. Chemical characteristics of clarified vinasse and F1 filtrate (average of three determinations).

In the filtrate F1 , resulted after the application of the process described above, it could be noted a significant reduction of the melanoidins content, humic acids and fulvic acids. The content of glycine-betaine remains high, and the ratio C:N is favorable for the action as fertilizer with nitrogen available for plants.

In the agro-fertilizer organo-mineral F2 were made determination of the content of humic acids, using the methods mentioned above. The content of humic acids-C, determined in the agro-fertilizer F2 is 15.7 g kg^"1, well above the content of humic acids in the initial vinasse, which demonstrates humification of melanoidins by steel slag / manganese and iron oxides present in the steel slag, according to the process presented above.

In the clarified vinasse and filtrate F1 was tested the phytotoxicity using bioassay of mung beans (Vigna radiata) seedlings. Seeds of V. radiata (cv. Pusa Bold) were sterilized into surface through repeated washes with sodium hypochlorite. The seeds were spread in sterile Petri plates 0 25 cm, on sterile filter paper, moistened with 10 ml of clarified vinasse or filtrate F1 . Petri plates were maintained at room temperature for 2 weeks, after which the numbered the leaves formed and was performed determinations of radicle / root and hypocotyl / stem length, wet and dry biomass. The results shown that, after application of the process describe about and after reduction of the content of soluble melanoidins, the initial phytotoxicity of clarified vinasse is significantly lowered - table 2.

Tab 2. Influence of applying clarified vinasse and filtrate F1 on mung beans ( Vigna radiata cv. Puss Bold) seedlings.

Determinations of the toxicity to aquatic organisms of clarified vinasse, filtrate F1 and a leachate of organo-mineral fertilizer F2 were done. Bioassays were performed with aquatic organisms, cnidaria Hydra attenuata, green algae Pseudokirchneriella subcapitata (known as Selenastrum capricornutum), and (micro) crustaceans Daphnia magna and Daphnia similis. For cultivation and (eco)toxicological determinations on P. subcapitata and H. attenuata were used the procedures described by OECD TG201 -2006/ISO 8692-2005 [ENV/J /MONO (2008) 28], and, respectively Trottier et al. 1997, Toxicol. Water qual. 12, 265-271 . For ecotoxicological determinations with D. magna and D. similis the used method was OECD TG202-2004/ISO 6341 -2005 [ENV/JM/MONO (2008)]. Toxicity was estimated by the modified Spearman-Karber method (Hamilton et al., 1977, Environ Sci. Tech. 11 : 714-719), using a function added to the Microsoft Office Excel 2010 (Microsoft, Redmont, WA, USA), and Tukey test of SAS/STAT software package 12.2. (Institute, Carry, NC, USA), to determine the significance level P < 0.01 . Various values of the EC50, LC50 and IC50 were obtained. For H. attenuata, effective concentration values (EC50) were calculated on the basis of morphological changes and any sub lethal effects on 50% of the population tested. For D. magna and D. similis, lethal concentration (LC50) was considered the concentration that causes the immobilization and lack of reaction of 50% of the population of micro-crustaceans on which the experiment was done. For P. subcapitata the inhibitory concentration (IC₅0), was calculated as the concentration that inhibits the growth of 50% of the tested population, compared to an untreated control group.

Leachate of organo-mineral fertilizer F2 was obtained by washing 100 g of fertilizer with 1 liter of deionized water, repeated five times for 5 min.

The results are presented in table 3 below.

Tab. 3. Value^* of IC₅₀, LC₅₀ and EC₅0 of clarified vinasse and of filtrate F1 for different aquatic organisms.

^*% of the growth media of the aquatic organisms.

The results demonstrate the effectiveness of the process proposed in reduction of the toxicity of melanoidinic compounds present in the initial vinasse. The toxicity that occurs in the case of the filtrate F1 is determined by pH of the growth medium reduction, and not to melanoidins. Apparently toxic effects which were observed in the case of leachate from organo-mineral fertilizers F2 are determined mainly by the lack of nutrient resulting from excessive dilution of the culture medium, and not as a result of potential run-off of toxic melanoidinic compounds.

The efficacy of fertilizer F1 as osmoprotectant which protect tomatoes plants against water stress was tested. Plants of tomato (Lycopersicum esculentum cv. Cristal F1 ), 60 days old transplants, were transplanted into pots of 25 cm diameter and 50 cm height, in which there were introduced 5 liters of growing substrate enriched with nutrients for the first few weeks of growth (Canna Terra Professional Plus, Canna International BV). Vegetation vessels were kept in greenhouse conditions at 22 ± 2°C during the day and 17±2°C during the night, with 12 hours photoperiod, supplemented with light intensity of 160 mcE/m²/s, derived from the halogen lamps, when light intensity decrease below 500 mcE/m² level. Experiment last for 60 days. The substrate contained reserves of nutrients, so the plants were fertilized only once, after 30 days from transplanting, by application of 55 ml of nutrient solution, 1 g/l of fertilizer 20-8-20 (N-P₂0₅-K₂0 Eurofertil TimacAgro, Romania). The experiment was conducted in randomized block with 4 repetitions for each treatment, every repetition including five plants. As a product of reference it has been used purified glicin-betaine (99%, Sigma Aldrich, St. Louis, Mo, USA), the experimental treatments including control treated only with water, water-stressed and non-stressed. The treatments involved the application of fertilizer 7.5% F1 fertilizer solution and of glycine-betaine 10 mM, 55 ml per kg of substrate (equivalent to 300 m³ / ha for soil horizons 0-45 cm), twice, initially as a growing substrate treatment, 5 days before transplanting, and as second treatment, after 30 days, through the inclusion into the mineral fertilizer nutrient solution 20-8-20 (N-P₂0₅-K₂0) as fertilizer solution 7.5% F1 and glycine-betaine 10 mM. The experimental treatments which where studied were:

V-i - control, not water stressed, treated with water; substrate initially treated with 55 ml of water per kg, eq. 300 m³/ha, fertigation with 55 ml solution of 1 g/l of mineral fertilizer 20-8-20 (N-P₂0₅-K₂0) per kg of substrate;

V₂ -control water stressed, treated with water, substrate initially treated with 55 ml of water per kg, eq. 300 m³/ha, fertigation with 55 ml solution of 1 g/l of mineral fertilizer 20-8-20 (N-P₂0₅-K₂0) per kg of substrate;

V₃ - not water stressed, treated with glicin-betaine, substrate initially treated with 55 ml 10 mM glicinbetaina solution per kg, fertigation with 55 ml solution of 1 g/l of mineral fertilizer 20-8-20 (N-P₂0s-K₂0) and glicinbetaina 10 mM, per kg substrate;

V₄ - water stressed, treated with glicin-betaine, substrate initially treated with 55 ml of solution per 10 mM glicinbetaina, fertigation with 55 ml solution of 1 g/l of mineral fertilizer 20-8-20 (N-P₂0₅-K₂0) and glicinbetaina 10 mM, per kg substrate;

V₅ - not water stressed, treated with F1 fertilizer, substrate initially treated with 55 ml solution 7.5% fertilizer F1 , fertigation with 55 ml solution of 1 g/l of mineral fertilizer 20-8-20 (N-P₂0₅-K₂0) and 7.5% fertilizer F1 , per kg of substrate; V₆ - water stressed, treated with F1 , substrate initially treated with 55 ml solution 7.5% fertilizer F1 , fertigation with 55 ml solution of 1 g l of mineral fertilizer 20-8-20 (N-P2O5-K₂O) and 7.5% fertilizer, per kg of substrate.

The water non stressed treatments were watered once every five days at 100% field capacity (55 ml per 1 kg of substrate), and water stressed treatments were watered each ten days at 100% capacity. At the end of the 60 days from transplanting date the plant were harvested, and the morphological parameters of plants, plant height, root length, leaves number and surface were determined. Data were processed by analysis of variance (Statistica 10, StatSoft, Tulsa, OK, USA).

The results are presented in table 4 below. Fertilizer F1 , obtained according to the above example of invention embodiment, applied as drench treatment of the soil, in the concentration of 7.5 percent, equiv. 1.23 g/l glycine-betaine, have an activity similar to the solution of purified 10 mM glycine-betaine, limiting the effect of the water stress effects on tomatoes plants and stimulating non-stressed plants.

Tab. 4. Influence of treatment with compositions made in accordance with the invention of the tomatoes plant, water stressed and not water stressed^*.

^* Values are followed by the same letter does not differ significantly P > 0.05; ** production per 30 day cycle of flowering-fructification

It was done also an experiment to test the efficacy of organo-mineral agro- fertilizer F2 on the nutrition of ornamental plants. The experiment was performed on cuttings of hybrid geranium (Pelargonium x hortorum) cv. "Moulin Rouge". Uniform cuttings, in term of stem and root development, were planted in plastic pots of 25 cm, containing a growth substrate enriched with nutrients (Canna Terra Professional Plus, Canna International BV). Pots were kept in greenhouse conditions at 22±2°C during the day and 17±2°C during the night, with 12 hours photoperiod, supplemented with light intensity of 160 mcE/m²/s, produced by halogen lamps when light intensity decrease below 500 mcE/m². Substrate contained initial nutrients reserve, and the plants were supplementary fertilized only after five weeks, applying 55 ml of nutrient solution, 1 g/l of fertilizer 20-8-20 (N-P₂O₅-K2O, Eurofertil, TimacAgro), per kg of substrate.

The additional fertilization treatments were the following:

\ - control, untreated with organo-mineral agro-fertilizer

V₂ -control, treated with a reference product, obtained by pelletizing in conditions similar, of a mixture of 2 parts of steel slag, 49 kg bio-char, and 52 kg of softwood sawdust, 4 g per kg of substrate, equivalent to 21 .6 tons/ha;

V₃ - treated with organo-mineral agro-fertilizer F2, 4 g per kg of substrate, equivalent of 21.6 tons/ha;

V₄ - treated with organo-mineral agro-fertilizer F2, 6 g per kg of substrate, equivalent to 32.4 tons/ha.

Each treatment included 12 pots, which were arranged in blocks of three per repetition, in a Latin square randomized schedule, 4 treatments in 4 repetitions. 12 weeks after transplanting of cuttings, the fresh and dry weight of roots and aerial parts of geranium plants were weighted. Data were processed by analysis of variance (Statistica 10, StatSoft, Tulsa, OK, USA).

Tab. 5. Effect of organo-mineral agro-fertilizer treatments on the growth of geranium plants.

The results presented in tab. 5 demonstrate an effect of growth stimulation of the ornamental plants of geranium, under the influence of treatments with organo- mineral fertilizer F2.

Claims

1. Process according to the invention characterized in that consist of the following steps: separation of an yeast concentrate from vinasse, which contains at least 10% yeast, by using a centrifugal separator, operated at min. x 8500 g, high pressure homogenization of yeast concentrate, homogenate refrigeration and maintenance of yeast homogenate to 4°C until further use; adjustment of the clarified vinasse pH to value 4, addition of steel slag, in a ratio of 2 parts steel slag to 98 parts vinasse, and maintaining at 60°C, with 20 rpm agitation and aeration with 0.2 liter air / liter medium/ min for 12 hours, in order to promote the catalyzation of the humification reaction, by the manganese and iron oxides present in steel slag; cooling of the mixture till 25°C, adjustment of pH in the mixture to 3.5, and addition of bio-char, in the ratio of 2 parts bio-char to 100 parts of vinasse - steel slag, and maintaining, with 20 rpm agitation, for 3 hours; adjustment of the pH of the mixture vinasse - steel slag - bio-char to 1.5 units, and maintaining at 25°C with 20 rpm agitation for 20 min, to complete the humic acids precipitation, pre-existing and formed by the catalytic action of iron and manganese oxides; filtration of the mixture of vinasse - steel slag - bio-char, on a rotative vacuum filter, with differential pressure of 0,6 MPa, with the separation of a filtrate (F), with high content of (glycine)betaine, usefull a nitrogen fertilizer F1 , and a precipitate (P); blending of the precipitate (P) retained on filter, with a residual moisture content of 20%, with bio-char, with a residual moisture content of 6%, with softwood sawdust, with residual moisture content of 14%, and homogenized yeast, with humidity of 85%, in the ratio of 6 parts precipitate (P) : 44 parts bio-char : 40 parts of softwood sawdust: 10 part homogenized yeast, and densification of the resulted mixture by compressing into a pellet press, with horizontal dyes, to form pellets with a length of approx. 15 mm and a diameter of 5 ... 8 mm, representing the organo-mineral agro-fertilizer F2.

2. Process according to claim 1 characterized in that the high pressure homogenization of yeast concentrate is done by passing through a high pressure piston homogenizer piston fitted with a knife edge valve, 2 cycles at 150 MPa and acidification to pH 2.0 of homogenate is done with phosphoric acid.

3. Process according to claim 1 characterized in that the steel slag used to catalyze the oxidation of fulvic acids and their coagulation as complexes of humic acids, contains at least 2.5% of manganese oxide and 25% iron oxides.

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4. Process according to claim 1 characterized in that the used biochar, with a specific microporous volume, W_a, of at least 0,6 cm³g^"\ and with a surface of micropores, S_mj_Cro, of min. 450 m g^"1, is obtained by micro-wave assisted pyrolysis, from a mixture of distiller grains, from bioethanol production, and corn stover, 25 ...50% distiller grain : 50 ... 75% corn stover, and micro-wave assisted pyrolysis is performed by exposing the above mixture for 20 minutes to micro-waves, with a power of 1000 W and an incident frequency of 2450 MHz.

5. Process according to claim 1 characterized in that densification of the mixture, precipitate (P) : bio-char : softwood sawdust : yeast homogenate, is performed with a specific power of 1 kW for 0.015 ...0.02 m² surface of the matrix, maintaining the temperature of the pelletizing mixture of about 65°C.

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