PL248922B1 - Method of producing biogel, biogel and use of biogel - Google Patents

Abstract

The subject of the application is a method for producing biogel using strains of environmental microorganisms isolated from samples of soil contaminated with hydrocarbons, which is characterized in that in the first stage, a liquid medium for the multiplication of microorganisms is prepared by heating 100 l of demineralized water in a mixer to a temperature of 20°C to 30°C, and subsequently, during uniform mixing, additives are introduced in quantities calculated in relation to their total mass, wherein casein hydrolysate is introduced first in an amount of 8% to 14% m/m and mixing is continued for not less than 15 minutes until the hydrolysate is completely dissolved, and then, while constantly stirring, from 0.08% to 0.085% m/m of sodium chloride, 0.15% m/m of iron (II) sulphate, 1.35% m/m of magnesium sulphate, 0.085% m/m of sodium sulphate are added. manganese (II), from 8.5% to 10% m/m of potassium dihydrogen phosphate, then glucose is added in an amount of 19.72% to 20% m/m and the composition is stabilized by stirring continuously for not less than 1 hour. +/- 5 min, and then sodium citrate is dosed in an amount from 56.4% to 60% m/m and all components are mixed until the mixture is completely homogenized, for not less than 1 hour, after which, in the medium thus obtained and maintained at a temperature of 25°C, a consortium of PW microorganisms composed of Pseudomonas, Enterococcus, Enterobacter strains or a consortium of PM microorganisms composed of Pseudomonas, Citrobacter, Legionella, Rhodococcus, Rahnella strains is subjected to the process of multiplication, whereby the multiplication continues until the biomass density of 109 [cfu/ml] is obtained, thus obtaining a liquid base, which is subsequently mixed with a gel base in the form of a gelatin gel with a concentration of 3% or an agar gel with a concentration of 3% or an agar gel with a concentration of 5%. in such a way that the gel base at a temperature of 40°C is poured into a mixer containing a liquid base at a temperature of 20°C to 25°C, stirring evenly and slowly until the bases are completely mixed, but for no longer than 15 minutes, after which the biogel thus obtained is packaged at room temperature from 18°C to 24°C, while the proportions of the liquid base to the gel base are 2:1. The subject of the application also covers the biogel obtained by the above method and its use.

Description

Description of the invention

The subject of the invention is a method for producing a biogel, particularly for remediating water and soil from hydrocarbon pollutants, and a biogel, particularly for remediating water and soil from hydrocarbon pollutants, as well as the use of a biogel, particularly using remediation plots.

It is known that the best soil and water purification results are achieved using microbiological preparations. For example, Russian patent application No. RU2013155969 (A) describes a biopreparation for the bioremediation of oil-contaminated soils, designed for the climatic conditions of the Far North. The biopreparation comprises a solid substrate carrier and a consortium of hydrocarbon-oxidizing microorganisms immobilized on its surface, containing the strains Bacillus vallismoris RNCIM B-11017, Exguobacterium mexicanum RNCIM B-11011, Serratia plymuthica VKM B-2819D, and Rhodococcus sp. VKM AC-2626D.

Another Russian patent application, No. RU2016150217 (A), discloses a preparation for biodegrading methanol from soil, ground, sea, freshwater, and saltwater. The preparation is made with the bacteria Bacillus megaterium VKPM B-607, Methylobacterium sp. VKPM B-5603, and Acinetobacter calcoaceticus VKPM B-3780 on a natural ion exchange sorbent. The method for preparing the preparation involves separate cultivation of the bacterial strains Bacillus megaterium VKPM B-607, Methylobacterium sp. VKPM B-5603, and Acinetobacter calcoaceticus VKPM B-3780 to a cell count of 10-10 mcl/ml. Subsequently, the culture fluids of the strains are mixed until the bacterial count reaches 10 mcl/ml and then sprayed onto the glauconite-containing sorbent as an aerosol. Capillary-chemical dehydration of the mixture is carried out by introducing air heated to 40°C. When the moisture content in the final biological product reaches 4%, the process is stopped.

Russian patent no. RU2149008 (C1) relates to a method for preparing a biopreparation, according to which an industrial strain of Bacillus subtilis is cultivated in a spore-supplying medium. The biomass is separated from the medium by mixing it with a stabilizing agent based on a sugar-gelatin medium with calcium glucanate in an amount of 0.1-5.0 wt.% or with acetylphthalylcellulose. The biomass and stabilizing agent are taken in a ratio of (1:1)-(1:1.5). The resulting mixture is dried and placed in gelatin capsules. The medium conditioner contains the following ingredients in g/l: potassium dihydrogen phosphate, 0.2-0.4; ammonium sulfate, 1.0-3.0; sodium citrate, 1.0-3.0; copper sulfate, 0.001-0.01; zinc sulfate, 0.002-0.005; iron sulfate, 0.0001-0.001; calcium chloride, 0.09-0.2; magnesium sulfate, 0.1-0.5; manganese sulfate, 0.01-0.1; peptone, 2.0-8.0; maltose, 1.0-5.0; water for balance.

Chinese patent application No. CN101177677 (A) discloses a method for producing microbial immobilized particles and is characterized in that a culture medium seed liquid is mixed with a gel liquid by weight of 5-20% for 12 to 14 hours at a temperature of 25-40° C. The immobilized particles are prepared using a granulator with a diameter of 1-4.00 mm, with a falling rate of 20 to 30 drops/min, and the particles are chemically crosslinked for 10 to 18 hours and then statically reinforced with a fixing agent for 23 to 58 hours. The mass ratio of the gel liquid is: sodium alginate (Na Alg) 2 to 3%, activated carbon (150 mesh), peat soil and/or fly ash 0.3 to 0.6%, and the balance is water. The bacterial culture medium is: Basal medium: beef extract 0.3%, peptone 1.0%, NaCl 0.5%, agar 1.5-2.0%, pH 7.0-7.2; Seed medium: glucose 1.25%, NH4NO 30.1%, MgSO4 7H2O 0.02%, KCI 0.02%, pH 7.0-7.2; Proliferation medium: glucose 4.0%, yeast extract 0.3%, KH2PO4 0.05%, MgSO4 7H2O 0.025%, (NH)2HPO4 0.2%, pH 7.0-7.2.

Japanese patent application No. JP2009017861 (A) concerns a method for producing polymer gel beads containing microorganisms, wherein the microorganisms and a water-soluble gel-forming polymer are suspended in an oil phase (rapeseed oil) to form a first emulsion, and the second emulsion is a solution containing polyvalent cations dispersed in the oil phase. Droplets of the dispersed phase collide with each other, forming gel beads containing the microorganisms.

U.S. Patent Application No. US2004101944 (A1) discloses microbial cultures for inducing microbiological processes in water bodies, soils, and sediments, comprising chemolithoautotrophic bacteria immobilized in a matrix in the form of capsules, gels, or gel beads. The matrix material is selected from a wide range of natural and synthetic polymers.

In another Russian patent application No. RU2565549 (A) a biopreparation is described containing a solid substrate carrier and a consortium of hydrocarbon-oxidizing microorganisms immobilized on its surface, containing the strains Bacillus vallismoris RNCIM B-11017, Exguobacterium mexicanum RNCIM B-11011, Serratia plymuthica VKM B-2819D, Rhodococcus sp. VKM AC-2626D and a mineral nutrient substrate for bacteria in the form of natural zeolite.

Japanese patent application JPS61247385 (A) relates to enabling the cultivation of a microbial cell in a gelatinous carrier such as alginate, agar, collagen, gelatin, polyacrylamide or a prepolymer.

Russian invention No. RU2678528 (A) discloses a preparation for biodegradation of methanol, including a compound of the bacteria Bacillus megaterium VKPM B-607, Methylobacterium sp. VKPM B-5603, Acinetobacter calcoaceticus VKPM B-3780 on a natural ion exchanger. The method of preparation comprises separate cultivation of the bacterial strains Bacillus megaterium VKPM B-607, Methylobacterium sp. VKPM B-5603 and Acinetobacter calcoaceticus VKPM B-3780 to a cell content of 10-10 mcl/ml. The culture fluids of the strains are mixed to a bacterial content of 10 ml/ml and then sprayed in the form of an aerosol onto a sorbent containing glauconite.

From Chinese patent application No. CN108018229 (A), a microbial hexavalent bacterial chromium removal agent is known, wherein a depositing agent and a microbial bacterial suspension are uniformly mixed according to a 1:1 ratio, and the obtained thick mixed liquid is dropped into a CaCl2 solution and is dispersed into beads.

A method for producing biological coal slag granules used for purifying polycyclic aromatic hydrocarbons in oil-contaminated soil is disclosed in Chinese patent application No. CN104651343 (A). The method is characterized in that it comprises the following steps: selecting high-yield hydrocarbon-degrading bacteria and inoculating an enriched liquid culture medium with high-yield degrading bacteria to obtain a turbid solution of degrading bacteria; then, a gel solution is prepared by taking pretreated coal slag as a carrier and modified PVA as a gel.

Another Chinese patent application No. CN 103275963 (A) discloses a method for producing microorganisms in the form of a microsphere for cleaning the river bed. The production method comprises the steps of: producing a PVA (polyvinyl alcohol) gel, comprising the step of preparing a PVA solution of 8-10% concentration, adding embedding agents to the PVA solution, e.g. sodium alginate of 0.5-2% concentration, calcium carbonate of 0.2-0.5% concentration, silicon dioxide of 2.0-4.0% concentration and attapulgite powder of 300-500 mesh or 300-500 mesh, activated carbon powder of 0.5-1.0% concentration and adding activated lime microorganisms of 10-15%. Forming the microspheres comprises the steps of adding PVA gel dropwise to a solution of boric acid saturated with calcium chloride; mixing; obtaining spherical particles activated with immobilized microorganisms with a particle size of 3-5 mm, carrying out cross-linking for 24-36 hours at 4-8°C and removing and washing with salt solution.

Polish patent application No. P.372636 concerns a method for obtaining a biopreparation for the degradation of diesel fuel hydrocarbons, which involves isolating strains of three bacterial species: Gordonia alkanivorans S7, Micrococcus luteus A.32.1, and Pseudomonas circulans Bp from an environment contaminated with petroleum hydrocarbons. These strains are then grown as a mixture in a shaken culture under aerobic conditions, at pH 6.5, at 28°C, for 72 hours, in a sterile medium containing an assimilable source of carbon, nitrogen, and phosphorus, growth substances, and diesel fuel. The resulting biopreparation is stabilized with sodium sulfide or optionally centrifuged and dried in a stream of sterile air.

Another Polish patent application, P.380007, discloses a soil bioremediation method involving the introduction of immobilized Yarrowia lipolytica yeast into the soil. The yeast is introduced directly into or around contaminated areas using the in situ method, in the form of a vaccine on an organic carrier such as sodium alginate, agar, gelatin, collagen, or bird feathers. The vaccine, in liquid or dry form, can be introduced in the form of granules, biofilm, or biogel, and the yeast constitutes 5 to 50% of the vaccine mass, and can also constitute 10 to 100% of the material introduced into the soil. The vaccine is introduced to a depth of 0.1 to 2 m into holes 0.1 to 1.0 m in diameter or into grooves 0.1 to 1.0 m wide, where their arrangement may be linear, transversely overlapping, or concentric, surrounding the area, or located at its lowest point, in the direction of groundwater flow, or, in the case of water body protection, near the shoreline beyond the reach of waves. The invention can be used in areas contaminated, in particular, by petroleum derivatives, waste from the oleochemical industry, vegetable and mineral oils.

In another Polish patent application no. P.403930 a method of bioremediation of soil contaminated with diesel oil is described, which consists in introducing into the soil to be purified a multiplied inoculum of bacteria of the genus Achromobacter, supported by vegetable oil, preferably rapeseed oil, and is characterized in that in the bioremediation process an inoculum of the bacterial strain Achromobacter xylosoxidans G21 isolated from soil contaminated with diesel oil is used, which is introduced into the contaminated soil and its cultivation is carried out, wherein the oxygenated vegetable oil is introduced into the environment to be purified in the period between 1 and 14 days of bioremediation and after the introduction of the oil the bioremediation process is continued under aerobic conditions.

The state of the art indicates that the most commonly used forms of biopreparations are liquids and suspensions, with powders, granules, or gel beads being significantly less common. Experience to date indicates that the greatest challenge with using liquid preparations and suspensions containing microorganisms is the application time after their production. These preparations can be optimally applied within several hours of production, with the microorganisms dying off within hours. This time frame prevents them from being considered a marketable product. Furthermore, conducting remediation processes using such preparations has significant limitations, not least because the production of microbiological preparations requires advanced technical resources and experienced specialists. Therefore, they are produced in the manufacturer's stationary laboratories and then transported to the application site. The survival of microorganisms between production and application is a key issue limiting the use of microbiological biopreparations in these forms. Liquid preparations are added to soil or water, and the full population of microorganisms is inoculated at once. Microorganisms enter the environment simultaneously, resulting in peak effectiveness initially and a gradual decline in effectiveness as the microorganisms in the environment die off. In a heavily polluted environment, the rapid introduction of microorganisms into it can significantly reduce the microbial population, or even completely kill the biological mass of the preparation.

It is also common knowledge that remediation processes using previously used preparations, primarily liquid preparations, are performed in designated remediation areas (plots). Traditional remediation plots or panels are initially subjected to the highest possible level of irrigation or even flushing. Contaminated soil is placed in piles and poured or sprinkled with liquid preparations, ensuring maximum saturation. High saturation of the piles is associated with the formation of leachate, which is collected by a drawdown system and filtered. Another important aspect is that conventional remediation panels require the development of an entire stationary infrastructure, including filtration and application systems. This entails not only formal procedures and lengthy permitting procedures, but also the high costs of constructing such a facility.

To limit the above-mentioned inconveniences, the inventors formulated an inventive problem consisting in the production of a microbiological preparation enabling effective bioremediation of soil and water from hydrocarbon pollutants in a form that allows its storage, transport and dosing.

The invention is aimed at a method of producing a biogel for remediation of soil and water from hydrocarbon pollutants; a biogel for remediation of water and soil from hydrocarbon pollutants and the use of the biogel, especially through remediation plots, wherein the biogel contains a consortium of active bacterial strains, immobilized and biologically stabilized in a gelling substance environment.

The method of producing biogel using strains of environmental microorganisms isolated from samples of soil contaminated with hydrocarbons is characterized in that in the first stage a liquid medium for the multiplication of microorganisms is prepared by heating 100 l of demineralized water in a mixer to a temperature of 20 to 30°C, then during uniform mixing additives are introduced in amounts calculated in relation to their total mass, with casein hydrolysate being introduced first in an amount of 8% to 14% m/m and mixing continued for no less than 15 min. until the hydrolysate is completely dissolved, and then, while stirring constantly, from 0.08% to 0.085% m/m of sodium chloride, 0.15% m/m of iron (II) sulphate, 1.35% m/m of magnesium sulphate, 0.085% m/m of manganese (II) sulphate, from 8.5% to 10% m/m of potassium dihydrogen phosphate are added, then glucose is added in an amount of from 19.72% to 20% m/m and, while stirring constantly, the composition is stabilized for not less than 1 hour. +/- 5 min., and then sodium citrate is dosed in an amount from 56.4% to 60% m/m and all components are mixed until the mixture is completely homogenized, for not less than an hour, after which, in the medium thus obtained and maintained at a temperature of 25°C, a consortium of PW microorganisms consisting of the following strains is subjected to the process of multiplication of microorganisms: Pseudomonas, Enterococcus, Enterobacter or a consortium of PM microorganisms consisting of the following strains: Pseudomonas, Citrobacter, Legionella, Rhodococcus, Rahnella, while the multiplication lasts until the biomass density of ¹⁰⁹ [cfu/ml] is obtained, thus obtaining a liquid base, which is subsequently mixed with a gel base in the form of a gelatin gel with a concentration of 3% or an agar gel with a concentration of 3% or an agar gel with a concentration of 5%. in such a way that the gel base at a temperature of 40°C is poured into a mixer containing a liquid base at a temperature of 20 to 25°C, stirring evenly and slowly until the bases are completely mixed, but not longer than 15 minutes, after which the biogel thus obtained is packaged at room temperature from 18 to 24°C, while the proportions of the liquid base to the gel base are 2:1.

The biogel produced by the above-mentioned method is used for the remediation of contaminated soils in-situ by introducing it into the soil through vertical and/or horizontal applicators, by gravity or mechanical means or by pressure injection, and for cleaning tanks or industrial separators by applying it to the tank walls with a roller, brush or spray.

Due to the intended bioactivity of the biogel according to the invention, it became necessary to select strains of environmental microorganisms capable of degrading hydrocarbons and surviving in the gel's low-water environment. Several hundred samples of soil contaminated with petroleum hydrocarbons were collected from various environments. Each sample was tested for contaminant content. From samples showing excessive hydrocarbon contaminant levels, naturally occurring soil microorganisms were selected. This provided a cross-section of various types of microorganisms from various environments and soils with different properties. To determine the preferred habitats of hydrocarbon-degrading microorganisms, aqueous solutions were prepared. Water extraction yielded samples to determine the microorganisms' habitats.

Table 1 contains tests revealing the number of microorganisms in the collected soil samples.

For each of the samples listed in Table 1, a pH test was performed 72 hours after extraction. The pH value is related to the impact of the preparation medium on the soil environment.

The pH level test results are presented in Table 2.

From the bacterial strains tested, identified in Table 2, sequencing was performed for strains exhibiting specific hydrocarbon degradation properties. Each sample was tested for hydrocarbon reduction using established methods. Among the samples containing microorganisms that caused the highest hydrocarbon reductions in the tested samples, the strains most effective for degrading various types of petroleum contaminants were selected and cataloged. Each sample was cultured, and based on morphological characteristics (shape, color, growth, colony appearance, etc.), over 100 bacterial strains with potential biodegradation properties were selected.

After their purification, several dozen strains were selected for surface tension analysis.

Table 3 presents the surface tension values of selected strains.

Based on morphological characteristics and surface tension values, strains with surface tension values lower than the surface tension of water and the culture medium were selected. DNA was isolated from the selected strains. The purified DNA samples were sequenced and identified.

Microorganisms from the following groups were identified:

- Acidovorax

- Acinetobacter

- Alcaligenes

- Bacillus

- Brevundimonas

- Candida

- Castellaniella

- Chromobacterium

- Citrobacter

- Comamonas

- Delftia

- Enterococcus

- Enterobacter

- Flavobacterium

- Lactobacillus

- Legionella

- Micrococcus

- Ottowia

- Proteus

- Providencia

- Pseudomonas

- Pseudoxanthomonas

- Ralstonia

- Radiobacter

- Rahnell

- Rodococcus

- Serratia

- Shinella

- Sphingobacterium

- Stenotrophomonas

- Ochrobactrum

- Vagococcus

All identified strains have been deposited in the microbanks of the Institute of Ecology of Industrial Areas and are part of a collection of microorganisms. All of them can be used (either as a consortium or individually) in various bioremediation processes in environments contaminated with petroleum hydrocarbons. From among the isolated strains, microorganisms (consortia) were selected based on their xenobiotic degradation properties, susceptibility to survival in a gel environment, and potential biosurfactant production.

The first consortium consists of the following strains: Pseudomonas, Enterococcus, and Enterobacter (designated PW). Numerous studies have confirmed that the best bioremediation results are achieved when used for the biodegradation of oils and gasoline.

The second consortium consists of strains of Pseudomonas, Citrobacter, Legionella, Rhodococcus, and Rahnella (designated PM). Numerous studies have confirmed that excellent bioremediation effects are achieved with its use in biodegrading various types of hydrocarbons.

As disclosed above, the selected consortia are soil bacterial consortia, previously isolated from soil samples, containing saprophytic, non-pathogenic microorganisms. Numerous previous studies have demonstrated that such a complex, multi-species biocenosis has a high adaptability to various environments and can colonize habitats with diverse living conditions.

The production of biogel with the participation of selected consortia involves the immobilization of microorganisms (PW or PM microorganism consortium) in gelling substances.

In the method according to the invention, gelatin and agar are used as gelling agents. Before inoculation, the consortium is grown in a liquid medium to a biomass density of approximately 109 [cfu/ml].

The standard proportions of the components have been determined for the amount of 100 l of demineralized water to which they are added and amount to a total of 2500 g to 3550 g, preferably 3020 g.

The quantities of individual components are indicated below in % by mass, calculated in relation to the total mass of additives used:

- from 8% to 14% m/m, preferably 11.6% m/m of casein hydrolysate;

- from 56.4% to 60% m/m, preferably 58% m/m of sodium citrate;

- from 19.72% to 20% m/m, preferably 19.85% m/m of glucose;

- from 0.08% to 0.085% m/m, preferably 0.082% m/m of sodium chloride;

- 0.15% m/m of iron (II) sulfate;

- 1.35% m/m of magnesium sulfate;

- 0.085% m/m of manganese (II) sulfate;

- from 8.5% to 10% m/m, preferably 8.95% m/m of potassium dihydrogen phosphate.

In the method according to the invention, 100 I of demineralized water is heated to a temperature of at least 20°C and not more than 30°C, after which the individual components calculated in mass amounts per 100% of the mass of all additives are poured sequentially, ensuring uniform, constant and slow mixing with a mixer speed of no more than 18 rpm and a temperature in the range of 20 to 30°C. First, casein hydrolysate is added to the demineralized water in an amount of 8% to 14% m/m, preferably 11.6% m/m and the whole is mixed until it is completely dissolved, while mixing is continued for no less than 15 min. Subsequently, from 0.08% to 0.085% m/m, preferably 0.082% m/m sodium chloride; 0.15% m/m iron (II) sulfate; 1.35% m/m magnesium sulfate; 0.085% m/m manganese (II) sulfate; from 8.5% to 10% m/m, preferably 8.95% m/m potassium dihydrogen phosphate, while maintaining uniform dosing to ensure better mixing of the components. The order of adding the components is arbitrary. The next component is added, glucose in an amount from 19.72% to 20% m/m, preferably 19.85 m/m. The composition is stabilized by maintaining uniform mixing for no less than 1 hour +/- 5 min, after which, with constant stirring, sodium citrate is uniformly dosed in an amount from 56.4% to 60% m/m, preferably 58% m/m. All components are mixed until complete homogenization of the mixture, for no less than 1 hour. The liquid medium, as a clear liquid, free of inclusions and visible turbidity, is ready for microorganism application. Until the inoculation process is complete, the mixture should be kept at 25°C with constant stirring.

Of course, by maintaining the indicated proportions of all components of the liquid medium in relation to 100 l of mineralized water, it is possible to produce correspondingly larger or smaller quantities in relation to a given amount of mineralized water.

Microorganisms are introduced into the prepared liquid medium using known methods. Microorganisms are multiplied sequentially to the required biogel production quantity, using multi-stage passaging. For each passaging step, a previously prepared liquid medium is used, into which an aqueous solution containing the microorganisms is poured. Multiplication occurs to a microorganism density of ¹⁰⁹ [cfu/ml], after which each step is passaged in a ratio no greater than 1:10 (for example, 1 liter is passed to 10 liters, 10 liters to 100 liters, etc.). The process in the liquid medium is continued until a biomass density of approximately ¹⁰⁹ [cfu/ml] is achieved. Finally, the liquid medium is obtained in the amount required to produce the desired amount of biogel. The liquid medium is maintained at a temperature of 20-25°C.

After each passage sequence, in order to confirm the safety of the biopreparation, an analysis is performed to exclude pathogenic microorganisms, e.g. Ercherichia coli, Salmonella sp., Pseudomonas aeruginosa and Enterococcus fecalis.

Next, the gel medium is prepared. Gelatin and agar-agar-based gels were developed, which, depending on availability and the needs of the remediation medium, can be used interchangeably. Gelatin is the most commonly used gelling agent. Agar is a plant-derived substance obtained from red algae, possessing gelling properties. It also serves as a neutral substrate for media used to cultivate bacteria. Its high porosity allows for the encapsulation of a large number of microorganisms in a relatively small volume. It exhibits strong hydrophilicity and resistance to physical and mechanical factors. It creates stable carriers with the additional advantage of not shrinking or swelling.

Numerous studies and trials have shown that the best bioremediation results (the best microorganism survival) are achieved using a 3% gelatin gel to produce the biogel. For the agar-agar-based gel, the best results were achieved with 3% and 5% gel concentrations. Higher concentrations of gelling agents (both gelatin and agar-agar) were also tested during the study, but all models were rejected due to the low microorganism survival in the gel.

Biogel is produced by combining a liquid base (liquid medium with implemented microorganisms) and a gel base in a 2 (liquid base) : 1 (gel base) ratio. Demineralized water is heated to 60°C in a mixer, and then the gel substance (agar or gelatin) is added. The gel base is prepared depending on the desired concentration (3% gelatin gel, 3% agar-agar gel, or 5% agar-agar gel). After adding the gelling agent, the mixture is mixed slowly and evenly until it is completely dissolved. The heating is then turned off and the gelling base is gradually cooled to 40°C. It is then poured into a mixer containing a liquid base at 20 to 25°C, stirring evenly and slowly until the bases are fully mixed, but for no longer than 15 minutes. After that, the biogel is immediately packaged in containers. Packaging is performed at room temperature, 18 to 24°C. After 24 hours, the biogel is ready for use.

The biogel produced by the method according to the invention is suitable for storage, the best storage temperature being 25°C.

The biogel according to the invention consists of a liquid base and a gel base in 2: 1 proportions, wherein the liquid base is a liquid medium with implemented consortia of microorganisms with a biomass density of approx. 109 [cfu/ml], and the gel base is 3% gelatin gel or 3% agar gel or 5% agar gel, and the liquid medium is a mixture of additives dissolved in demineralized water, calculated with respect to 100 I of water and containing the following components calculated in relation to their total amount: from 8% to 14% m/m, preferably 11.6% m/m of casein hydrolysate; from 56.4% to 60% m/m, preferably 58% m/m of sodium citrate; from 19.72% to 20% m/m, preferably 19.85% m/m of glucose; from 0.08% to 0.085% w/w, preferably 0.082% w/w sodium chloride; 0.15% w/w iron (II) sulfate; 1.35% w/w magnesium sulfate; 0.085% w/w manganese (II) sulfate; from 8.5% to 10% w/w, preferably 8.95% w/w potassium dihydrogen phosphate. The microorganism consortia are the "PW" consortium - composed of the following strains: Pseudomonas, Enterococcus, Enterobacter and "PM" - composed of the following strains: Pseudomonas, Citrobacter, Legionella, Rhodococcus, Rahnella. Biogel with the above-indicated composition is used for the remediation of contaminated soils and for cleaning industrial tanks or separators.

In-gel viability tests were performed for 3% and 5% gelatin gels and 3% and 5% agar-agar gels. The results presented in Tables 4 to 7 are averaged for the "PW" and "PM" consortia.

Table 4 - results for 3% gelatin gel

Table 5 - results for 5% gelatin gel

Table 6 - results for a 3% Agar-Agar based gel

Table 7 - results for a 5% Agar-Agar based gel

The results confirm that the biogel of the invention guarantees the preservation of the microorganisms' viability, maintaining their full viability during a period that allows for free transport without significant restrictions in terms of transport conditions or time. The studies demonstrated that the viability of the microorganisms remained virtually unchanged for up to three months.

Room temperature conditions are sufficient to ensure a long shelf life for microorganisms in remediation. Furthermore, any deviations from optimal conditions will not cause bioremediation properties to change as rapidly as with liquid preparations, as the biogel acts as an environmental buffer for the microorganisms. The gel form ensures a "closed microbiological environment," meaning the microorganisms, being separated from the environment, are less susceptible to any environmental changes.

Additional features of biogel, resulting from its formula, include the ability to create a layer separating pollutants from the surrounding environment. In the case of pollutants that cause emissions, such as evaporation, a biogel layer can be applied, which mechanically separates the pollutant from the environment, creating an active barrier. This barrier will limit or even eliminate the pollutant's impact on the environment while also acting directly on the pollutant, neutralizing or degrading it. It creates a kind of active compress, enclosing the pollutant degradation process in a closed, separated space between the substrate and the biogel compress.

Example - biogel test according to the invention

The aim of the test is to demonstrate the disintegration time of biogels in a gel environment, periods of temperature and pH increase, as key indicators of microorganism activity.

pH - is associated with the impact of biogels, or more precisely, its components, on the soil, the decomposition of which causes a temporary increase in pH during the decomposition of the substrate.

Temperature is an indicator of the xenobiotic decomposition process as a result of microbial activity. Microorganisms, through natural metabolic processes, break down carbon bonds, using carbon to obtain the energy they need for survival. As a result of their activity, energy is produced and emitted into the environment in the form of thermal energy. Monitoring temperature increases can be used as an indicator of the duration and intensity of hydrocarbon degradation occurring in the earth.

The test was performed using a biogel with both consortia (liquid base for the PW consortium and subsequently for the PM consortium with microorganisms multiplied to a density of ¹⁰⁹ [cfu/ml], produced in accordance with the method of the invention and taking into account its process conditions, where the liquid medium consisted of: 100 I of demineralized water, 380 g of casein hydrolysate; 1750 g of sodium citrate; 610 g of glucose; 2.6 g of sodium chloride; 5 g of iron (II) sulfate; 40 g of magnesium sulfate; 2 g of manganese (II) sulfate; 270 g of potassium dihydrogen phosphate, while the gel base was a 3% gelatin gel and a 3% agar-agar gel, respectively. Biogel was produced in accordance with the process conditions of the method according to the invention by mixing the liquid base as above with 51 kg of gel base.

The produced biogels were compared to the reference test, which is a liquid base with a microorganism density of ¹⁰⁹ [cfu/ml].

The direct goal of the test was to determine the microorganism release time, their duration of action, and the dynamics of the processes. Other data was also collected during the test, which indirectly also aimed to indicate the difference in the gel's performance compared to the liquid preparation in terms of hydrocarbon reduction efficiency and the measurement of emissions associated with the biogel's operation.

The soil was prepared for testing. Measured quantities of contaminated soil were formed into heaps (so-called remediation plots). Before the soil came into contact with the native soil, a layer of a tight geomembrane was applied, ensuring the separation of all processes taking place in the plot from the native soil. Test sequences were prepared, each consisting of 10 trials, i.e., 8 test trials (4 produced biogels with PW and 4 with PM, respectively) and 2 reference trials (1 liquid base with PW and 1 with PM, respectively). Hydrocarbon sensors were placed above each test plot (remediation plot), collecting data on emissions related to the conducted process. Each test began with a pH and temperature measurement, as the starting parameters for each test sequence. Each test was described and labeled according to the dose and type of biogel administered. After the biogel was administered, battery-powered pH and temperature measuring devices were placed in the test samples and left for further observations. pH and temperature observations were conducted daily to verify that the process was proceeding correctly. The aim of the tests was to demonstrate hydrocarbon degradation depending on the type of biogel, soil, and doses, therefore, pH and temperature measurements constituted ongoing monitoring. Graphs were generated based on the test results.

Graph 1 - pH changes of biogel from the PW consortium and 3% gelatin gel vs. liquid base from PW

Graph 2 - pH changes of biogel from PW consortium and 3% Agar gel vs. liquid base from PW

Graph 3 - pH changes of biogel with PM consortium and 3% gelatin gel vs. liquid base with PM

Graph 4 - pH changes of biogel with PM consortium and 3% Agar gel vs. liquid base with PM

Graph 5 - temperature changes biogel with the PW consortium and 3% gelatin gel vs. liquid base with PW Graph 6 - temperature changes biogel with the PW consortium and 3% Agar gel vs. liquid base with PW Graph 7 - temperature changes biogel with the PM consortium and 3% gelatin gel vs. liquid base with PM Graph 8 - temperature changes biogel with the PM consortium and 3% Agar gel vs. liquid base with PM The tests conducted characterize the mode of action of the biogel, its extended action time compared to the liquid preparation, and confirm the direct relationship between hydrocarbon degradation processes and the release of microorganisms from the gel medium to the environment.

The biodegradation activity of the bacterial preparations stabilized in gels was then verified. Analyses were performed in triplicate for two biogel variants: PW + 3% agar-agar gel; PW + 3% gelatin gel; and PM + 3% agar-agar gel; and PM + 3% gelatin gel. Before testing, the soil was analyzed to determine the initial concentrations of the tested substances. The experiment was conducted at room temperature. Biodegradation tests were conducted in two 60-day cycles. Before gas chromatography, the samples were thoroughly mixed to homogenize the sample. Examples of gas chromatography results are presented in the tables. The test results were related to the dry mass of the soil. The dry mass of the soil samples was determined according to the PN-75/C-04573/10 standard.

Table 8 - biodegradation activity - PW variant + 3% agar-agar gel

Table 9 - biodegradation activity - PW variant + 3% gelatin gel

Table 10 - biodegradation activity - PM variant + 3% agar-agar gel

Table 11 - biodegradation activity - PM variant + 3% gelatin gel

The biogel according to the invention guarantees the preservation of the microorganisms' viability, maintaining their full viability for a period of time that allows for free transport without significant restrictions in terms of transport conditions and time. Studies have shown that viability remained virtually unchanged for a period of three months, guaranteeing high quality of the preparation, surpassing all liquid preparations. The gel form provides a "closed microbiological environment," meaning the microorganisms, while not in direct contact with the environment, are less susceptible to any environmental changes. Because the microorganisms are protected from sudden environmental changes, they are not susceptible to the shock associated with sudden environmental changes during application.

Research conducted during the trials confirmed that the biogel applied to the remediated environment demonstrated 100% population effectiveness of the microorganisms contained within, with no loss in numbers from production to application. The microorganisms are maintained in a viable form throughout the entire cycle, eliminating the losses associated with their "waking up" as is the case with all dry forms.

As seen in the graphs, a unique feature of the biogel according to the invention is the gradual and consistent release of microorganisms into the environment after application. After application, the microorganisms remain within the gel environment, which is subject to degradation. This method releases microorganisms into the environment over a defined period of time, systematically and steadily, resulting in a continuous application to the soil (or other substrate) throughout the entire degradation of the gel substrate, which can last from 3 days to several weeks after application. The biogel formulation therefore eliminates the risk of microbial shock after application, eliminates the risk of microbial population decline, and ensures the product's high performance over a long period, fully utilizing its full potential. The product's duration of action allows for less frequent applications, resulting in less labor and significant cost reductions. Biogel also produces lower emissions, both in terms of odor emissions and leachate.

Of particular note is the use of a remediation plot constructed as a pile of contaminated soil with a tight geomembrane substrate. This plot is not an installation (often necessary when using remediation fluids), does not require complicated construction procedures, and is inexpensive and quick to install. The incoming soil can undergo bioremediation immediately in the plot, eliminating the need for storage and securing it during storage. Tests and studies have therefore revealed that it is possible to locate the plots near the soil undergoing remediation, minimizing emissions associated with its transportation.

By using a remediation plot in the tests, there were no emissions related to factors such as evaporation, and odor emissions from the bioremediation process were low. The plot did not generate any process waste that would have to be disposed of for further disposal. Using a remediation plot with the biogel according to the invention provides an effective, simple, and relatively inexpensive remediation field, which was previously impossible without generating leachate and requiring construction permits and related procedures. It eliminated the cost risks associated with obtaining waste processing permits, minimized emissions, and ensured a quick and safe remediation process as close as possible to the site of need.

The biogel according to the invention can be used for various applications

Application 1 – In-situ remediation of contaminated soil and water, with particular emphasis on the remediation plot described above. This application is the intended application of the invention. Biogel can be introduced into the soil through vertical and/or horizontal applicators, using gravity or mechanical means, including pressure injection. Biogel introduced into the applicators does not cause a sudden and simultaneous dispersal of microorganisms and liquid across the ground, but releases microorganisms (and other substances) gradually over a given period of time. Biogel application does not introduce large amounts of liquid into the ground, so it does not disturb the groundwater level or cause soil leaching. Biogel releases microorganisms in a targeted, localized manner, without being susceptible to more arbitrary spread than liquids. Microorganisms released from the gel environment have a significantly higher survival rate and effectiveness, and the duration of the preparation is significantly longer than that of liquid preparations.

Application 2 - Cleaning Industrial Tanks: Biogel can be used in industry, where it can be used for tank cleaning. One example of this is cleaning gas station tanks. Fuels precipitate various types of sediments that remain on the internal walls of the tanks. Furthermore, temperature changes in tanks cause condensation to form, so the tank is never completely empty, leaving condensation and other heavier fractions separated from the fuel at the bottom. During inspection and cleaning, the tank is emptied, but sediments remain on the walls, and contaminated condensation, fuel residue, and bottom sediments remain at the bottom. Biogel, as a hydrocarbon neutralizer, allows for the neutralization of petroleum substances directly in the tank. Due to its form, it can be applied to the tank walls with a roller, brush, or spray, without the need for personnel to enter the tank. The latter method can completely limit activities that generate the highest risk of ignition, harmful working conditions for people, and the highest emissions of vapors into the atmosphere.

Application 3 - Industrial Tank Cleaning: Another application with enormous market potential is separator cleaning. Oil separators are found everywhere with vehicular traffic. They are installed in residential areas with parking lots, parking lots, maneuvering areas, gas stations, mechanical workshops, fuel unloading stations at industrial facilities, along highways, and anywhere else where there is a risk of oil-based substances being released into the environment. Biogel can be used to clean separators similarly to its use in gas station tanks, although the number of separators used, both in industry and in everyday applications, is significantly greater. Biogel can be sprayed onto walls and/or used to neutralize sediment in separator clarifiers.

Claims (10)

Patent claims 1. A method for producing biogel using strains of environmental microorganisms isolated from samples of soil contaminated with hydrocarbons, characterized in that in the first stage a liquid medium for the multiplication of microorganisms is prepared by heating 100 l of demineralized water in a mixer to a temperature of 20 to 30°C, and subsequently, during uniform mixing, additives are introduced in amounts calculated in relation to their total mass, wherein casein hydrolysate is introduced first in an amount of 8% to 14% m/m and mixing is continued for not less than 15 min. until the hydrolysate is completely dissolved, and then, while stirring continuously, from 0.08% to 0.085% m/m of sodium chloride, 0.15% m/m of iron (II) sulphate, 1.35% m/m of magnesium sulphate, 0.085% m/m of manganese (II) sulphate, from 8.5% to 10% m/m of potassium dihydrogen phosphate are added, then glucose is added in an amount of from 19.72% to 20% m/m and, while stirring continuously, the composition is stabilized for not less than 1 hour. +/- 5 min, and then sodium citrate is dosed in an amount from 56.4% to 60% m/m and all components are mixed until the mixture is completely homogenized, for not less than 1 hour, after which, in the medium thus obtained and maintained at a temperature of 25°C, a consortium of PW microorganisms composed of the following strains is subjected to the process of multiplication of microorganisms: Pseudomonas, Enterococcus, Enterobacter or a consortium of PM microorganisms composed of the following strains: Pseudomonas, Citrobacter, Legionella, Rhodococcus, Rahnella, while the multiplication lasts until the biomass density of 109 [cfu/ml] is obtained, thus obtaining a liquid base, which is subsequently mixed with a gel base in the form of a gelatin gel with a concentration of 3% or an agar gel with a concentration of 3% or an agar gel with a concentration of 5% in such a way that the gel base at a temperature of 40°C is poured into a mixer containing a liquid base at a temperature of 20 to 25°C, stirring evenly and slowly until the bases are completely mixed, but not longer than 15 minutes, after which the biogel thus obtained is packaged at room temperature from 18 to 24°C, while the proportions of the liquid base to the gel base are 2:1. 2. The method according to claim 1, characterized in that the production of the liquid medium takes place at a stirrer speed of no more than 18 rpm, at a temperature in the range of 20 to 30°C. 3. The method according to claim 1 or 2, characterized in that 11.6% m/m of casein hydrolysate is added. 4. A method according to claim 1, 2 or 3, characterized in that 0.082% m/m of sodium chloride is added. 5. The method according to any one of the preceding claims 1 to 4, characterized in that 8.95% m/m of potassium dihydrogen phosphate is added. 6. The method according to any one of the preceding claims 1 to 5, characterized in that 19.85 m/m of glucose is added. 7. A method according to any one of the preceding claims 1 to 6, characterized in that 58% m/m of sodium citrate is added. 8. Biogel containing consortia of microorganisms, characterized in that it consists of a liquid base and a gel base in 2:1 proportions, wherein the liquid base is a liquid medium with implemented PW or PM microorganism consortia with a biomass density of ¹⁰⁹ [cfu/ml], and the gel base is 3% gelatin gel or 3% agar gel or 5% agar gel, and the liquid medium is a mixture of additives dissolved in demineralized water, calculated with respect to 100 I of water and containing components calculated with respect to their total mass in the form of: casein hydrolysate in an amount from 8% to 14% m/m, preferably 11.6% m/m; sodium citrate in an amount from 56.4% to 60% m/m, preferably 58% m/m; glucose in an amount from 19.72% to 20% m/m, preferably 19.85% m/m; sodium chloride in an amount from 0.08% to 0.085% m/m, preferably 0.082% m/m; iron (II) sulphate in an amount of 0.15% m/m; magnesium sulphate in an amount of 1.35% m/m; manganese (II) sulphate in an amount of 0.085% m/m and potassium dihydrogen phosphate in an amount from 8.5% to 10% m/m, preferably 8.95% m/m, while the PW consortium includes the following strains: Pseudomonas, Enterococcus, Enterobacter, and the PM consortium includes the following strains: Pseudomonas, Citrobacter, Legionella, Rhodococcus, Rahnella. 9. Use of the biogel defined in claim 8 for the remediation of contaminated soils in-situ by introducing it into the soil through vertical and/or horizontal applicators, in a gravity or mechanical manner, or by pressure injection. 10. Use of the biogel defined in claim 8 for cleaning tanks or industrial separators by applying it to the tank walls with a roller, brush or spray.