RU2405636C2 - Method of obtaining biomass of microorganisms, with biological recycling of organic compounds in purified medium - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention can be used in biological treatment of water, soil and similar media from organic compound contaminants of arbitrary composition and concentration, as well as when recycling organic production wastes. The method involves determination of content of organic compounds in the medium to be treated, selection of native microorganisms isolated from the treated medium, addition of the obtained mass to the treated medium with addition of macro-elements: nitrogen, phosphorus, potassium, magnesium, sulphur and trace elements: iron, zinc, manganese, molybdenum, cobalt, boron and control of physical and chemical characteristics of the treated medium. Microorganisms are selected based on the highest growth rate of biomass. Content of oxygen in the treated medium is kept between 2·10^-3 and 1.4 kg/m³ and pH is kept between 5 and 7.5. Macro- and trace elements are added based on their presence in the treated medium in total amount of (1-8)·10^-3 kg for macro-elements and 1.1·10^-7-1·10^-5 kg for trace elements per 1 MJ of complete energy of oxidation of the recycled organic compounds in an oxygen medium.

EFFECT: method enables detoxification of contaminating organic compounds to a maximum admissible concentration level and their conversion to a state which is suitable for use.

16 ex

Description

The invention relates to methods for producing nutrients from environmental pollutants and chemical wastes and can be used in the biological treatment of water, soil and similar media, as well as in the disposal of industrial wastes, for example, used automobile oils.

The prior art there are many methods of biological treatment of the environment from pollutants, for example, the method of biological disposal of oil pollution according to US Pat. RF №2076150, publ. 03/27/1997, in which individual saprophytic strains are used as cultures, a method for biological cleaning of the soil from pollution with oil and oil products according to US Pat. RF №2191643, publ. 10/27/2002, suitable for purification from aromatic hydrocarbons of high concentration, for example, from oil and oil products, in the presence of salts of heavy metals.

At the same time, it is known to use organic substances in biotechnological processes as substrates for growing microorganisms in order to obtain products of their vital activity. The substances used include carbohydrates, hydrocarbons, alcohols, proteins and others. Examples include the synthesis of acetic acid by Acetebacter and Gluconobacter strains using ethyl alcohol as a carbon and energy source, and the preparation of citric acid using sucrose or starch. Known protein preparations paprin and haprin are obtained using strains of the genus Candida from n-paraffins C ₁ -C ₃₀ . Many other biotechnological products (antibiotics, vitamins, amino acids, food additives, etc.) are obtained by processing biogenic carbohydrates: mono-, oligo and polysaccharides. Sometimes technogenic organic wastes are used, for example, cyclohexine in the production of lysine, indole in tryptophan biosynthesis [Biotechnology, ed. A.A. Baleva. M. 1988; Elina N.P. The basics of biotechnology. St. Petersburg, 1995; Kefeli V.I., Dmitrieva G.A. Biotechnology. Pushchino, 1989; Egorova T.A., Klunova S.N. Zhivukhina E.A. The basics of biotechnology. M. 2003].

There is a method of disposing animal and bird waste according to the patent of the Russian Federation No. 2067570, publ. 05/09/1999. Primary biomass is grown on these wastes, which, in turn, serves as a substrate for growing a secondary group of microorganisms, which are used as protein and vitamin feed for animals and poultry. The composition of the substrate is adjusted by the carbon-nitrogen ratio by the addition of a lignin hydrolyzer.

A common disadvantage of the known methods is the limited list of processed media represented by oil and biogenic products, while environmental pollutants, and especially toxic industrial wastes, can have an arbitrary composition both by the nature of their constituent substances and by their quantitative ratio. The methods do not include obtaining useful products, or their list is limited. The efficiency of the known methods of disposal is low, since utilizing microorganisms do not go through the selection stage, only carbon and nitrogen are taken into account from the components of the processed medium, while other macro- and microelements vital for the activity of microorganisms are not taken into account.

Closest to the claimed technical solution is a method of biological treatment of various environments from organic substances and their mixtures (Method of biological treatment of soil and water from organic compounds of aliphatic, carbocyclic and heterocyclic series and their mixtures of arbitrary composition. Patent of the Russian Federation No. 2315670, publ. January 27, 2008 adopted as a prototype). In the known method, polluting organic compounds (pollutants) are subjected to biological utilization to the level of maximum permissible concentration of MPC, providing physico-chemical characteristics of the processed medium that are favorable for microflora life. Organic compounds turn into cellular substances of utilizing microorganisms, water and carbon dioxide, which are included in the general cycle of the substance cycle in the biosphere. The remains of microorganisms under the influence of regulatory trophic mechanisms is included in the cycle of natural microbiocenoses, part of them under the influence of saprophytic microflora turns into safe humic substances.

The disadvantages of the prototype.

The material and energy potential of utilized organic compounds in the prototype method is not used rationally. The material potential is understood as the mass of chemical elements that goes into the biomass of microorganisms, and the energy potential is the amount of energy that can be released during the complete oxidation of organic compounds with oxygen. The recycling process decays exponentially as power supplies are consumed, and the final biomass of utilizing microorganisms tends to a minimum so as not to upset the ecological balance of the natural environment.

When implementing the prototype method, the components of pollutants and components of additives are irretrievably lost and cannot be used to obtain useful products. Polluting organic compounds are utilized to carbon dioxide and water, other chemical elements form forms of low commercial value: carbon, which is part of organic substances, is converted to carbon dioxide, hydrogen to water, sulfur to sulfates, bound nitrogen to gaseous elemental nitrogen.

According to the known method, in the process of biological disposal of polluting organic compounds, utilizing microorganisms go through the reproduction stage, however, when the power sources disappear, they die, their concentration gradually tends to the background (natural). In the prototype method, the possibility of regulating the final biomass of microorganisms is mentioned, however, there are signs that minimize their final content to prevent secondary (biological) pollution. According to the prototype method, the material and energy potential of a mixture of organic substances is available for a limited list of biological cultures (several hundred types of utilizing microorganisms), but is not available for most strains of producing microorganisms, as well as for other life forms (zooplankton, insects, fish, birds) . The composition of the resulting biomass of utilizing microorganisms is not controlled, and this biomass cannot be used as a nutrient substrate for microorganisms producing useful products.

At the same time, the biomass of utilizing microorganisms (at the peak of its content) is of great value as an object of biological or technical transformation or other use. Organic compounds processed by microorganisms are usually present in soil, water and similar media in the form of chemical pollutants, but chemical waste products should also be included here. When setting the objective of the invention, organic substances that have penetrated into the environment and are in concentrations in excess of the MPC are classified as pollutants, and organic substances excluded or inaccessible to the technological process are among the waste products. Typically, such waste is accumulated in sedimentation tanks or at toxic landfills and stored in conditions isolated from the natural environment at concentrations several orders of magnitude higher than the MPC. In both cases, for the purposes of the claimed invention, soil, water, and similar media containing organic pollutants, as well as waste directly or mixtures thereof, act as a processing facility and are collectively referred to as the processing medium, and the qualitative composition of this medium and the concentration of organic compounds are not limited.

The aim of the invention is a more rational use of organic substances that make up the processed environment, providing useful products.

An object of the invention is the biological detoxification of polluting organic compounds and their mixtures to the MPC level with the conversion of these substances into a form suitable for useful use. In other words, the task is to transform the physical mass of contaminants into the biomass of microorganisms, which can be isolated and used either directly as a product or raw material, or as a nutrient substrate for a wide range of biosystems.

The problem is solved in that in the method for the biological utilization of organic compounds and their arbitrary mixtures, including determining the content of organic compounds in the processed medium, selection of native microorganisms, introducing them into the processed medium with the addition of macro- and microelements, and regulating the physicochemical characteristics of the processed medium microorganisms additionally select on the basis of the highest rate of biomass growth, the number of activating macros introduced About - and microelements are regulated relative to the energy of polluting organic compounds, and the process of biological disposal is carried out in the mode of increasing the final biomass of the mentioned microorganisms. To do this, provide the total content in the processed medium of nitrogen, phosphorus, potassium, magnesium, sulfur in the range (1-8) · 10 ^-3 kg per 1 MJ of energy of organic compounds, taking into account their presence in the medium, the total quantitative content of iron, zinc, manganese , molybdenum, cobalt, boron - in the range of 1.1-10 ^-7 -1 · 10 ^-5 kg per 1 MJ of energy of organic compounds, taking into account their presence in the environment. By controlling the process, the concentration of polluting organic compounds is reduced to the MPC level, after which the accumulated biomass is used as a valuable biological product, namely, it is either left in the processed medium and used as fertilizer, or it is isolated and used as biotechnological raw material or as a multipurpose substrate for growing microorganisms producing useful products.

As indicated above, the aforementioned energy potential of pollutants refers to the amount of energy released when they are completely oxidized in an oxygen atmosphere, i.e. when converting carbon to CO ₂ , hydrogen to H ₂ O, sulfur to SO ₄ , etc.

The method according to the invention consists of the following steps: a) selection of microorganisms; b) their introduction into the processed environment; c) introduction of activating substances; d) creating optimal conditions for growing biomass of microorganisms; e) use of the resulting biomass.

Features of the invention common to the prototype:

- indigenous microorganisms are used for selection;

- when selecting microorganisms, a consortium is distinguished that can intensively utilize the target (available) organic compounds;

- microorganisms contribute to the processed medium in an amount of 0.001 to 30 kg / m ³ ;

- nitrogen, phosphorus, potassium, magnesium and sulfur are used as activating additives;

- adjust the physico-chemical parameters of the environment in which microorganisms work.

When solving the technical problem, they provide a mode of biomass growth up to the moment of utilization process termination. To do this, use the essential features that distinguish the claimed invention from the prototype:

- microorganisms additionally select on the basis of the rate of biomass synthesis and select the most productive consortium;

- the quantitative content of macro- and microelements is linked to the energy potential of the processed organic compounds;

- macrocells are added at the rate of (1-8) · 10 ^-3 kg per 1 MJ of energy of polluting organic compounds;

- trace elements are added at the rate of 1.1 · 10 ^-7 -1 · 10 ^-5 kg per 1 MJ of energy of polluting organic compounds;

- the accumulated biomass of utilizing microorganisms is used as a biotechnological product.

Data on the energy potential of pollutants needed to calculate the amount of additives within the stated limits are contained in chemical reference books (see, for example, “Brief Chemical Reference Book” edited by V. A. Rabinovich. Publishing House “Chemistry.” L., 1977 )

The above conditions are necessary and sufficient for the continuous increase in the biomass of utilizing microorganisms from the moment of introduction to the moment of disposal of polluting organic compounds. However, in relation to specific technical problems, some features may be specified.

In particular, when utilizing pollution to the MPC level, biomass accumulation is more effective if the following conditions are met:

- when the ratio of macronutrients is within the limits of N: P: K: Mg: S = (6-14) :( 1-3) :( 1-3) :( 0.01-0.1) :( 0.05-0 , 2);

- when the ratio of trace elements within Fe: Zn: Mn: Mo: Co: B = (1-5) :( 0.1-2.2) :( 0.1-2.4) :( 0.1-0 6) :( 0.01-0.4) :( 0.01-0.3);

- when the oxygen content in the medium is in the range from 2 · 10 ^-3 to 1.4 kg / m ³ ;

- when the concentration of salts of alkali and alkaline earth metals in the processed medium is not higher than 380 kg / m ³ ;

- when the concentration of salts of heavy metals in the processed medium is not higher than 180 kg / m ³ ;

- in some cases, the concentration of salts of heavy metals to the desired level can be reduced by electrolysis;

- if the electrolysis products inhibit the process of reproduction of microorganisms, the electrodes can be isolated by membranes;

- if the concentration of organic compounds is too high (for example, chemical waste), a neutral filler (for example, sand) can be added for convenience.

The obtained biomass of utilizing microorganisms can be used, in particular, for the isolation of heavy metals, the production of biogas, riboflavin, lysine, bound nitrogen, and plant protection products. In addition, biomass can be left in the soil as nitrogen or other fertilizers and trace elements, as well as isolated and used as a food source for multicellular organisms and animals, or burned to conserve carbon.

The selection of indigenous microorganisms after sampling is carried out in three stages as follows. At the first stage, microorganisms that are capable of assimilating processed organic compounds are isolated on solid media. Such species are always present in a polluted environment. At the second stage, from a selected colony of microorganisms on solid media, inoculation is carried out for periodic cultivation. The nutrient medium is prepared in the laboratory using recyclable organic compounds and activating mineral components. For further work, only those species that give the greatest biomass increase in terms of 1 MJ of energy of organic compounds are left, for which selection is carried out according to two indicators: according to the rate of reproduction and productivity of the biomass. The latter indicator depends on the viability of the species and environmental conditions. Selection is carried out using a flow-through cultivation system operating in a chemostat mode. The chemostat fermenter is seeded with cultures isolated in the second step. Gradually increasing the flow rate of the nutrient medium and controlling the concentration of organisms, isolate cultures that combine a high reproduction rate with high bio-productivity. Microorganisms unsuitable for solving the problem are washed out of their system. As a result of selection, a consortium unique to the problem being solved is obtained, which is propagated in the required quantity.

In practice, as micro- and macro-elements are included in biomass over time t, a steady decrease in the concentration of activating additives as mandatory building components for an increase in biological mass m occurs. Due to this, the indicator dm / dt may tend to zero or take a negative value, as occurs in the prototype method. Based on the laws of metabolic processes and microbiological experiments, we determined and introduced into the claims the ranges of the total amount of additives and the intervals of their relative content, providing dm / dt> 0 and the highest biomass yield. Theoretically, for 1 MJ of energy contained in the utilized organic compound, no more than 3.4 · 10 ^-2 kg of dry biomass can be obtained at the output. It has been experimentally established that, with the found ranges of acidity, oxygen content and activating additives, a stable yield of biomass of microorganisms is ensured in the amount of (1.1-3.4) · 10 ^-2 kg / MJ, or 30-100% of the possible in terms of absolute dry weight. Part of the energy potential of organics in the process of biosynthesis passes into heat, part of the material potential in forms that cannot be used, and the specified range characterizes the efficiency and efficiency of the process.

Due to the physicochemical characteristics of some organic compounds, their disposal occurs at a low rate of reproduction of microorganisms. At low values of the specific rate of biomass growth, namely, when the number of cells doubles for about 70 hours, the material and energy potential of substances is not spent on biomass accumulation, but on maintaining the life of cellular organisms, which reduces the overall economic coefficient for carbon and energy sources. In addition, in the processed medium there are always any inhibitory factors that should be eliminated or adjusted, therefore, for the proper use of the method, it is necessary to take into account general information on microbiology. To increase the final biomass yield, the medium is optimized according to such indicators as the content of macro- and microelements per unit energy potential of the organic matter, the concentration of salts of alkali, alkaline earth and heavy metals, mass oxygen content, and acidity. We have experimentally established that for a fast and stable increase in biomass it is necessary to maintain the oxygen content in the reaction zone from 2-10 ^-3 to 1.4 kg / m ³ at an acidity of pH 5-7.5. Sources of macro-micronutrients can be not only external, but also internal. When activating additives are added in the amounts and ratios indicated above, account must be taken of their content in the processed medium. The temperature is chosen above zero, mainly 15-35 ° C. If you deviate from these parameters, the biochemical composition of the produced biomass becomes depleted, unbalanced, and to use it as a universal substrate, additional correction is required, which reduces the economic effect of the application of the method.

The method is as follows:

a) cultivate the required consortium of utilizing microorganisms; b) introduce it into the processed medium; c) make activating additives in an amount determined by the energy potential of the processed organics; d) adjust the physicochemical characteristics of the medium; f) microorganisms are grown in the biomass accumulation mode. The process is completed with a decrease in the concentration of organic compounds to the MPC level. A further increase in biomass is negligible, and after a certain time, the biomass begins to decrease. At this point, utilizing microorganisms can be isolated from the medium being cleaned. The use of such an indicator as the specific energy potential of organic compounds in MJ / kg makes it possible to dose the required amount of activating additives and calculate the gross yield of biomass. The following methods are used to isolate the biomass of microorganisms from a liquid medium. a) Isolation by centrifugation. The purified volume of fluid is centrifuged at 5000-6000g for 10-15 minutes. 90-95% of biomass is released. b) Isolation by flotation. A flotation agent that moistens the surface of the cells is selected. After air purging, the biomass is concentrated in the resulting foam. c) Isolation by electrophoresis. If the cells of microorganisms have an electric charge, then when creating an electric potential sufficient to move microorganisms, the biomass is concentrated at the electrode of the corresponding polarity. d) Isolation by precipitation using gelling agents. The method is used, in particular, for the purification of drinking water from suspended particles. Al ₂ (SO ₄ ) ₃ is added to the medium containing microorganisms in doses of 10 ^-2 - 1 kg per 1 m ³ . A gelatinous precipitate forms, which carries microorganisms to the bottom. d) Isolation by the method of changing the density gradient. Raw biomass has a specific gravity of 1.05-1.10 t / m ³ . As the density of the surrounding medium increases due to the addition of soluble salts, the biomass floats and collects in a floating layer. f) Isolation by filtration. Bacterial filters are used to separate biomass from the medium being cleaned.

To isolate microorganisms from a solid medium (soil, soil, filler), it is diluted with water to a fluid state, mineral components are precipitated, then one of the above methods or a combination thereof is used.

Example 1 illustrates the procedure for taking into account the energy potential when calculating the amount of activating additives and biomass yield. There are 4 tons of a mixture of organic compounds: acetone, diesel, isoamyl acetate, toluene, 1 ton each. In the directories we find the total value of the energy potential of the listed components (10 ³ MJ): acetone 31.5; diesel fuel 44; isoamyl acetate 33.5; toluene 42.5. Only 151.5 · 10 ³ MJ. According to the invention, the mass of macroadditives will be from 151.5 · 10 ³ MJ · (1 · 10 ^-3 ) kg / MJ to 151.5 · 10 ³ MJ · (8 · 10 ^-3 ) kg / MJ, or from 0.151 to 1 , 21 t, trace mineral mass is negligible. Theoretically, the entire mass of organics and additives (4 t + 1.21 t) can be transferred to the biomass of microorganisms. In this example, the yield of dry biomass, taking into account losses, will be 151.5 · (1.1 · 10 ^-2 -3.4 · 10 ^-2 ) t, or from 1.67 to 5.15 tons. With a different amount of activating additives or their absence, the process is unstable, the biomass yield is less than the calculated one, the efficiency of the method is low.

The following example demonstrates the bioavailability of the energy and material potential of utilized organic compounds for microorganisms producing a useful product.

Example 2. It is required to obtain a plant protection product (presowing disinfection of seeds of grain crops) based on Trichoderma harzianum mushrooms. There is an aqueous solution with a total volume of 5 m ³ with an arbitrary content of polluting compounds (kg / m ³ ): phenol 1.3; dioxane 0.6; aniline 0.4; thiophene 0.21. Representatives of the species T. harzianum are not able to absorb the listed substances. For this reason, according to the invention, a consortium of utilizing microorganisms is used, capable of assimilating these substances, and their biomass is used to produce a useful product.

Solution: at the first stage, selected utilizing microorganisms, mineral salts NH ₄ NO ₃ , (NH ₄ ) ₂ HPO ₄ , KCl, Na ₂ SO ₄ , MgCl ₂ and trace elements in the calculated amount were added to the water mixture. The biomass grown on a mixture of organic compounds was isolated from the medium using a centrifuge. Since fungi of the T. harzianum species have high enzymatic activity and are capable of assimilating biopolymers, one can limit oneself to the surface disintegration of utilizing microorganisms with destruction of cell membranes. Wet biomass with a water content of 70% was subjected to heat treatment in an autoclave at 110 ° C for 1 hour. The thermally degraded biomass was diluted with sterile water in a ratio of 1: 9. After introducing the inoculum at a dose of 0.5 g per 1 liter of substrate, T. harzianum was grown under conditions of deep aeration of 100 liters of air per 1 liter of medium per hour at a temperature of 25 ° C and a pH of 6.5. The process lasts 120 hours. The analysis showed that the substrate from the biomass of utilizers contains the entire set of food sources necessary for the propagation of fungi of the species T. harzianum and does not need to introduce any additional substances.

The following example describes an approach in which some useful products are highlighted and some are used in a recycled environment.

Example 3. The task is to obtain useful products when cleaning the soil from petrochemical production wastes. Pollution is represented by sulfate acids, hydrocarbons and toluene in the ratio (kg / t) 16: 11: 0.1. Solution: a mixture of organic compounds is recycled as described above with the participation of mesophilic microorganisms. After reducing the concentration of pollutants to the MPC level, a soil layer saturated with microorganisms is treated under anaerobic conditions, for example, in a tank. Mesophyllum microorganisms acting as utilizers are subjected to enzymatic lysis at a temperature of 50-60 ° C, thermophilic methane-forming microorganisms are introduced, and the obtained lysis products become the subject of methane fermentation. Waste gases consist of 90-95% of methane, carbon dioxide and nitrogen. The resulting biogas can be used as fuel or for organic synthesis. The resulting solid residue is a source of nitrogen, phosphorus, potassium, magnesium and trace elements and can be used as organic fertilizer to increase soil fertility.

Example 4 shows how, without the use of expensive equipment, to accumulate the working volume of microorganisms for the disposal of large volumes of polluting organic compounds or waste. To carry out large-scale work - cleaning the area of 18 hectares from pollutants - a large amount of microbiological culture is required. The territory is contaminated with oil - 120 kg per 1 ton of soil, as well as drilling waste. Composition of drilling waste: saipan, gabroyl, carboxymethyl cellulose, polypac, superflux, ecopack, prasstol, clear. The total content of these compounds is 1.6 kg per 1 ton of soil. The soil is also contaminated with produced water with a salt content of 18 kg per 1 ton of soil. Solution: in the laboratory, a consortium was obtained that can utilize oil and drilling additives at a specified salinity level. By the method of periodic cultivation for 8 days, 1 kg of culture was grown. Then, a microbiological plantation in the form of soil beds was prepared at the place of work and conditions were created for the propagation of microorganisms in the soil on the plantation. The beds in a certain mode are watered with culture, mineral fertilizers, oil, a solution of drilling additives, formation water. The process is quantified using analytical equipment. The biomass doubling time was 8-12 hours. After 4 days, a mixture was obtained with a concentration of microorganisms of 50 kg / m ³ , i.e. 20 m ^{3 of} soil contains more than 1000 kg of biomass, which is enough to carry out cleaning work on the territory.

Examples 5-9 characterize the preparation of various useful products from a single mixture of organic compounds.

Example 5. Obtaining bound nitrogen. A liquid mixture of arbitrary organic compounds was used in the experiment: 1.6 kg of isoamyl acetate is contained in 1 m ^{3 of} water; furan 0.23 kg; resorcinol 0.18 kg; toluene 0.11 kg; diesel fuel 0.09 kg. Selected microorganisms are introduced into the mixture — nitrogen fixers that absorb these substances in a dose of 0.11 kg / m ³ . Activating additives are also added that do not contain nitrogen and ensure the physiological balance of nitrogen fixers: a source of phosphorus - NaH ₂ PO ₄ , a source of potassium - KCl, a source of magnesium - MgCl ₂ , a source of sulfur - Na ₂ SO ₄ , trace elements Fe, Zn, Mn, Mo, In, Co in the ratio of the features of the invention. The process of utilization of pollutants to the MPC level occurs under conditions of aeration with an oxygen-containing gas at a pH of 5.8-7.2. The result is a non-toxic solution containing a large amount of bound nitrogen, both in dissolved form and in the protein-nucleic complex of cells. After biomass disintegration, it can be used as a nitrogen-containing source of nutrients, for example, during hydroponic plant cultivation.

Example 6. Obtaining lysine. A consortium of microorganisms, selected on the organic compounds indicated in Example 5, is introduced into the water system at a dose of 0.2 kg / m ³ . The sources N, P, K, Mg, S are introduced. The grown biomass is separated from the culture fluid. After acid hydrolysis and neutralization of the excess acid, a culture is cultured that produces lysine, for example Brevibacterium flavum. Since microorganisms of the Brevibacterium flavum species have insufficient lytic activity, they carry out such disintegration that depolymerization of biopolymers to the level of mono- and oligomers is achieved, after which the nutrient medium becomes readily available to microorganisms producing lysine. The use of microbial biomass as a substrate is advantageous in that it is not necessary to introduce macro- and microelements and growth factors, in this case biotin, into the nutrient medium, since they are contained in sufficient quantities in hydrolysis products.

Example 7. Obtaining riboflavin - vitamin B ₂ . The biomass of utilizing microorganisms grown in Example 5 is subjected to temperature degradation during autoclaving. The resulting substrate is seeded with a producer of riboflavin, for example Bacillus subtilis. The accumulation of riboflavin proceeds in a known manner. In this system, there is also no need to introduce growth factors and mineral components.

Example 8. Obtaining biogas. The biomass grown in Example 5 is placed under anaerobic conditions with a temperature optimum of 50-60 ° C, where a methane-forming thermophilic culture, for example, Metanospilum, is bred. The resulting methane is used, for example, as fuel.

Example 9. The reservoir is contaminated with a mixture of organic compounds of the same composition as in examples 5-8, however, the concentration of substances is a thousand times lower. After biological utilization, the extraction of biomass of utilizing microorganisms is not profitable due to their low concentration. For this reason, zooplankton has been introduced into the reservoir. The object of its nutrition is utilizing microorganisms, and zooplankton itself is food for invertebrate larvae and fish. Thus, in the purified reservoir, a useful food chain is formed.

From examples 5-8 it is seen that the same mixture of organic substances or the like can act as a nutrient substrate for the production of a wide variety of biotechnological products. The reverse phenomenon is also possible - obtaining identical products from various sources. It should be noted that the substrate obtained from the biomass of utilizing microorganisms is available for processing with conventional enzyme preparations, without the participation of cell systems.

Nitrogen-fixing microorganisms are able to utilize almost all organic compounds, with the exception of halide-containing ones. However, the presence of halide-containing substances in the medium does not interfere with the nitrogen fixation process. At the same time, the energy potential of polluting organic substances is used to convert atmospheric nitrogen into a bound form assimilated by plants.

Example 10 explains the complex application of the method for cleaning the environment to obtain a useful product and demonstrates the provision of bioavailability of polluting organic compounds for plants. It is required to clean the soil contaminated with organic substances and increase the content of nitrogen absorbed by plants in it. Contamination example: meta-cresol 0.8 kg; phenol 1.2 kg; naphthalene 0.4 kg; DDT 0.22 kg per 1 ton of soil. These contaminants do not contain nitrogen, and DDT is a highly toxic halogen-containing compound. Solution: nitrogen-fixing microorganisms that absorb cresol, phenol and naphthalene are selected and introduced into the cleaned medium in the amount of 4 · 10 ^-2 kg per 1 ton of cleaned soil. In addition, DDT destructive microorganisms are selected and introduced into the medium to be cleaned in an amount of 2 · 10 ^-2 kg per 1 ton of the soil to be cleaned. For 1 ton of soil add 1 kg of superphosphate, 1 kg of potassium chloride, 0.1 kg of magnesium chloride, 0.1 kg of sodium sulfate, 0.01 kg of compounds containing Fe, Zn, Mn, Mo, Co, B. In the process of disposal organic compounds with nitrogen fixers for each 1 MJ of pollutant energy synthesized 6 · 10 ^-3 kg of bound nitrogen in terms of NH ₄ ion. Since the biodegradation of DDT proceeds in stages, with the formation of fragments that do not contain halogens, the energy potential of DDT is partially used by nitrogen-fixing microorganisms. In turn, the nitrogen formed in the soil provides the need for DDT destructive microorganisms in this element. However, in this process, an ammonium form of nitrogen is formed, which is unstable and has high volatility. To fix nitrogen, a nitrofixer is added to the medium to be cleaned - a biotechnological strain that converts unstable ion (+ NH ₄ ) to stable (-NO ₃ ). At the end of the process, the soil is not only cleaned, but enriched with sources of bound nitrogen available for woody-grass vegetation. As experiments have shown, the yield of grain crops increased by 10% compared with the control, which fully pays off biotechnological operations.

Examples 11-13 illustrate the organization of useful food bioshemes.

Example 11. Ensuring the availability of polluting organic compounds for zoological objects. Gray forest soil is contaminated with a mixture of the following organic compounds: naphthylamine, pyran, rhizocine, aviation kerosene. The content of substances is 0.32; 0.46; 2.1; 1.3 kg per 1 ton of soil, respectively. After microbiological destruction into the soil saturated with the biomass of microorganisms, earthworms were launched, in the amount of 1000 individuals per 1 m ³ . Feeding on microorganisms, the worms multiplied and increased their biomass. Earthworms, in turn, are eaten by birds and rodents. Consequently, the material and energy potential of polluting organic substances is converted into a form accessible to zoological objects.

Example 12. The formation of species composition in ecological systems. The soil is contaminated, for example, with fuel oil, o-xylene and phenol. Concentration of pollutants at 0.8 kg per 1 ton of soil. According to the features of the invention, utilization microorganisms (in this case, nodule nitrogen fixers), macro- and microelements, with the exception of the nitrogen source, are introduced. The territory is seeded with a mixture of seeds of plants related to cereals, cruciferous and legumes. Since nodule nitrogen fixers mainly concentrate on the root system of legumes, this allowed legumes to become dominant plants in this ecological system. In this particular case, utilizing microorganisms convert polluting organic compounds into a nitrogen source for legumes.

Example 13. Regulation of the biocenosis and the organization of a useful food biochem. Water in two closed reservoirs is polluted with dioxane, ethylene glycol monomethyl ether, and benzoic acid. The content of each substance is 10 ^-1 kg per 1 m ³ . The native microflora was selected as described above. Utilization of organic compounds was carried out in two oxygen modes: in the first reservoir with an oxygen content of 6 · 10 ^-3 kg per 1 m ³ , in the second reservoir with an oxygen content of 2 · 10 ^-2 kg per 1 m ³ . The utilization of pollutants proceeds identically, however, with different oxygen contents in the declared range, the species composition of secondary biocenoses is different. With a minimum oxygen content, the food chain forms microorganisms - invertebrates - amphibians. With a moderate oxygen content of 2 · 10 ^-2 kg per 1 m ³ it becomes enough to breed fish, so instead of amphibians, fish become the dominant vertebrates in this biocenosis.

Example 14. The conservation of carbon.

One reason for global warming is the annual release of several billion tons of carbon dioxide into the atmosphere. The decomposition of polluting organic substances and toxic waste contributes. It is useful to reduce the intensity of the flow of CO ₂ into the atmosphere, which is achieved by carbon conservation. For this, biomass grown on a mixture of organic compounds according to any of the above examples is subjected to thermal degradation, for example, heating to 600 ° C. Biomass decomposes to form elemental carbon (soot), water, nitrogen, and mineral salts. The carbon obtained in this way can be stored indefinitely, i.e. canned and removed from the circulation of CO ₂ in nature.

Example 15. Obtaining biomass in the presence of salts of heavy metals. Utilization of organic compounds, such as alcohols and aldehydes of aliphatic alcohols, while increasing the organic mass for nutrient substrates is sometimes difficult due to the high concentration of heavy metals. The content of heavy metal salts of 180 kg / m ³ and higher inhibits the process. The use of heavy metal precipitating agents is not always justified, since toxic bulk sediments include part of the grown biomass, which leads to the loss of valuable biological raw materials. For this reason, it is advisable to use electrolysis of the medium. Electrolysis is carried out by a constant or pulsating current, using electrodes from a biologically neutral and water-insoluble material, such as coal, graphite. By changing the voltage in the circuit, heavy metals are sequentially deposited on the cathode, starting with the metal having the lowest reduction potential. The growth of biomass can stop if the restoration leads to the formation of substances toxic to microorganisms, for example, chlorine, bromine, etc. In this case, the corresponding electrode is isolated from the main volume of the processed medium, for example, the electrode is enclosed in a semipermeable membrane.

When some heavy metals are transferred to the biomass of microorganisms, the field of application of the resulting nutrient substrates is limited due to their toxicity. To clean biomass, it is disintegrated at boiling point. Chemically stable complexes of heavy metals with bioorganic substances break down cells by disintegration in an acidic environment and converting heavy metals into water-soluble salts. Then the acids are neutralized and the multipurpose substrate is removed from the heavy metals by the electrolytic method. Obviously, with a sufficiently high content of heavy metals, the claimed method is economically justified for the production of heavy metals, mostly rare.

Example 16. In water, glucose, glycerin, ethanol of 0.5 kg per 1 m ³ with the presence of copper sulfate 5 kg / m ³ , zinc sulfate 22 kg / m ^{3 are} dissolved. It is necessary to process organic compounds into biomass, transfer biomass to a substrate and clean it of heavy metals. Solution: utilization microorganisms, activating additives are placed in the tank, provide aeration with air or oxygen, and regulate acidity. After completion, the disposal of organic compounds to the MPC level, the biomass of microorganisms is separated by centrifugation. To the biomass add 1% solution of sulfuric acid in relation to 9 volumes of a solution of H ₂ SO ₄ per 1 volume of biomass. The mixture is heated for 30 minutes at 100 ° C, then the acid is neutralized with a NaOH solution to pH 7. Coal electrodes are introduced and a voltage of 2V is applied at a current of 25A. Zinc and copper are deposited at the cathode. After reducing the concentration of zinc and copper to the level of MPC, the nutrient substrate is used to produce a useful product.

Thus, to solve the technical problem, two conditions are combined: a decrease in the concentration of organic compounds to the MPC level and the yield of the maximum amount of biomass. The biomass of utilizing microorganisms grown on utilized organic compounds can be used in at least three ways. Firstly, it can be left in the soil or water in the form of fertilizer components that increase its fertility or productivity. Secondly, once isolated, biomass can be used as a universal biological substrate for the life of most microorganisms producing. In this case, a biocycle is organized in which the mass and energy of pollutants are transformed into the biomass of intermediaries, i.e. microorganisms utilizers, which, in turn, is used to power the microorganisms producing. A useful product is obtained at the output, for example, riboflavin, methane, bound nitrogen, etc. Finally, the biomass of utilizing microorganisms is suitable for direct technological use, for example, for fermentation, burning for carbon conservation, growing zooplankton, in the form of animal feed, etc.

The advantages of the method for producing and using the biomass of microorganisms according to the invention. Due to its versatility, the claimed method has economic, environmental and social aspects. The problem is solved not only to utilize organic compounds in water, CO ₂ , heat, as in the analogues and prototype, but to completely translate the material and energy potential of harmful / useless organic compounds into a form suitable for further use. The method provides the conversion of any toxic organic compounds, including in the presence of heavy metals, into the biomass of microorganisms, followed by its translation into a universal substrate suitable for growing producer microorganisms. In addition to the synthesis of useful biotechnological products, the method allows to reduce carbon dioxide emissions into the atmosphere, to obtain heavy metals or biofuels from biomass. It is possible to transfer the biomass of microorganisms into the biomass of another biological form suitable for feeding animals (fish, birds), i.e. The method allows you to create the desired environmental relationships between species, organize useful food biochemistry.

Information sources

1. Biotechnology // Ed. A.A. Baleva. M., 1988.

2. RF patent No. 2067570, publ. 05/09/1999.

3. RF patent No. 2315670, publ. 01/27/2008 (prototype).

Claims (5)

1. A method of producing biomass of microorganisms in the biological utilization of organic compounds in a cleaned medium, including determining the content of organic compounds in the cleaned medium, selection of native microorganisms isolated from the cleaned medium, introducing the resulting consortium into the cleaned medium with the addition of macroelements: nitrogen, phosphorus, potassium, magnesium sulfur and trace elements: iron, zinc, manganese, molybdenum, cobalt, boron and regulation of physico-chemical characteristics of the processed medium, characterized in that the selection of microorganisms is carried out on the basis of the highest growth rate of biomass, the oxygen content in the cleaned medium is maintained from 2 · 10 ^-3 to 1.4 kg / m ³ , and the pH of the cleaned medium is maintained from 5 to 7.5, and macro- and microelements contribute taking into account their presence in the cleaned medium in a total amount of (1-8) · 10 ^-3 kg for macrocells and 1.1 · 10 ^-7 -1 · 10 ^-5 kg for microelements per 1 MJ of the total oxidation energy of utilized organic compounds in the medium oxygen, while receiving biomass of nitrogen-fixing microorganisms, nitrogen is not introduced. 2. The method according to claim 1, characterized in that the relative mass ratio of said macronutrients is selected within the range of N: P: K: Mg: S = (6-14) :( 1-3) :( 1-3) :( 0 , 01-0.1) :( 0.05-0.2), the relative mass ratio of the mentioned trace elements is chosen within the limits of Fe: Zn: Mn: Mo: Co: B = (1-5) :( 0.1-2 , 2) :( 0.1-2.4) :( 0.1-0.6) :( 0.01-0.4) :( 0.01-0.3). 3. The method according to claim 1, characterized in that the salinity of the medium to be purified with salts of alkali and alkaline earth metals is not higher than 380 kg / m ³ and the salinity of salts of heavy metals is not higher than 180 kg / m ³ . 4. The method according to claim 3, characterized in that the concentration of salts of heavy metals in the cleaned environment is reduced by electrolysis. 5. The method according to claim 4, characterized in that the waste microorganisms are protected from electrolysis products by surrounding the electrodes with semipermeable membranes.