SK279941B6 - Process for preparing a mixture of microorganisms binding nitrogen from the air, increasing phosphorous compounds solubility and decomposing oil sediments - Google Patents
Process for preparing a mixture of microorganisms binding nitrogen from the air, increasing phosphorous compounds solubility and decomposing oil sediments Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/80—Separation, elimination or disposal of harmful substances during the treatment
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/141—Feedstock
- Y02P20/145—Feedstock the feedstock being materials of biological origin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
Description
Vynález sa týka spôsobu prípravy zmesi mikroorganizmov na bakteriálne viazanie dusíka zo vzduchu, na zvyšovanie rozpustnosti vo vode nerozpustného fosforu obsiahnutého v pôde a na bakteriálny rozklad nespracovateľného kalu v oleji vznikajúceho pri výrobe jedlých olejov. Vynález je významný z hľadiska ochrany životného prostredia pomocou pôsobenia metabiózy mikroorganizmov.The invention relates to a process for the preparation of a mixture of microorganisms for the bacterial binding of nitrogen from air, for increasing the solubility of the water-insoluble phosphorus contained in the soil, and for the bacterial decomposition of unprocessed sludge in the oil resulting from edible oil production. The invention is important in terms of environmental protection by the action of metabiosis of microorganisms.
Doterajší stav technikyBACKGROUND OF THE INVENTION
V niekoľkých posledných desaťročiach sa v poľnohospodárskej výrobe používali prevažne dusíkaté umelé hnojivá. Dosahovali satým veľké výnosy úrod. Stále používanie umelých hnojív, ak to nebolo v spojení s použitím maštaľného hnojiva, spôsobilo, že sa pôda vplyvom veľkého množstva chemikálií stala kyslá a mikrobiálny život v pôde bol potlačený, pôda sa zhutňovala, zhoršili sa vodné a vzdušné podmienky pôdy. Okrem toho do spodných vôd prenikal škodlivý dusičnan. Pri veľkých dažďoch vodný prúd odplavil granulované umelé hnojivo do potokov a takto sa dostával dusičnan aj do vodných tokov. Jednorazovým dávkovaním veľkého množstva dusíkatého umelého hnojiva rastliny, najmä záliiadué rastliny, prijímali nadmerné množstvo dusičnanu, ktorý negatívne pôsobí na biologický organizmus, najmä zvlášť škodlivo pôsobí dusičnan na dojčatá a malé deti, lebo v krvi vyvoláva methemoglobinémiu, ktorá môže byť aj smrteľná. Preto sa zaviedlo sledovanie obsahu dusičnanov najmä v zelenine a určila sa ich horná prípustná hranica. Ale v praxi sa veľmi ťažko uskutočňuje stála kontrola obsahu dusičnanov v žiaducom širokom rozsahu.In the last few decades, mainly nitrogen fertilizers have been used in agricultural production. They achieved great yields for the Satan. The continued use of artificial fertilizers, if not combined with the use of livestock fertilizers, caused the soil to become acidic due to the large amount of chemicals, and the microbial life in the soil was suppressed, the soil compacted, soil water and air conditions deteriorated. In addition, harmful nitrate has penetrated the groundwater. During heavy rains, the water stream washed away the granulated fertilizer into the streams, and thus the nitrate also entered the watercourses. By a single dose of large amounts of nitrogenous fertilizer, the plants, in particular backbone plants, have received excessive amounts of nitrate, which have a negative effect on the biological organism, especially nitrate, especially on infants and young children, causing methemoglobinemia in the blood. Monitoring of nitrate levels has therefore been introduced, particularly in vegetables, and their upper acceptable limit has been established. However, in practice, it is very difficult to carry out a constant control of the nitrate content in the desired wide range.
Preto sa dostáva stále viac do popredia praktická možnosť bakteriálneho viazania dusíka zo vzduchu. Podporne pôsobí aj problém zvyšovania cien dusíkatých umelých hnojív v nadväznosti na zvyšovanie cien energií z dôvodu, že výroba dusíkatých umelých hnojív vyžaduje veľké množstvo energie. Po objavení bakteriáhieho viazania vzdušného dusíka sa zvyšuje úsilie o využitie dusíka zo vzduchu vo veľkom rozsahu v pôdnom hospodárstve v poľnohospodárskej praxi, najmä v poslednom období.Therefore, the practical possibility of bacterial binding of nitrogen from the air is becoming increasingly important. The problem of the increase in the price of nitrogenous fertilizers in connection with the increase in energy prices is also supportive, because the production of nitrogenous fertilizers requires a large amount of energy. Following the discovery of bacterial binding of air nitrogen, efforts are being made to use nitrogen from air to a large extent in land management in agricultural practice, especially in recent years.
Známe je viazanie dusíka baktériami rodu Rhizóbium, ktoré na koreňoch vikokvetých rastlín, napr. fazule, sóji, ďateliny vytvárajú hľuzy, ktoré viažu dusík zo vzduchu. Toto viazanie dusíka je však ohraničené len pre oblasť vikokvetých rastlín.Known is the binding of nitrogen by bacteria of the genus Rhizobium, which on the roots of flowering plants, e.g. beans, soybeans, clover create tubers that bind nitrogen from the air. However, this nitrogen bonding is limited only to the flowering plant region.
Podstata vynálezuSUMMARY OF THE INVENTION
Uvedené nevýhody do značnej miery odstraňuje spôsob prípravy zmesi na viazanie dusíka podľa vynálezu, ktorého podstata spočíva v spôsobe využitia niektorých draho v baktérií rodurízotoňacŕer, ktoré sú schopné viazať dusík zo vzduchu tým, že v priebehu ich množenia v pôde, počas ich životnosti, vydiftindujú nimi syntetizované formy dusíka do pôdy ako dusičnany, dusitany, amónium, aminokyseliny, bielkoviny s malými molekulami, ktoré sa viažu na organickú hmotu pôdy odkiaľ ich korene rastlín vstrebávajú. Takto priebežne vznikajúce dusíkaté látky sa nevymývajú z pôdy a neznečisťujú prostredie. Veľkou výhodou je, že počas vegetatívneho obdobia je dusík rastlinám dodávaný kontinuálne a harmonicky. Vhodnými druhmi sú bakteriálny kmeň Azotobacter croococcum CCM 4642 a bakteriálny kmeň Azospirillium brasiliense CCM 4644. Tieto bakteriálne kmene boli uložené v autentizovanej Českej zbierke mikroorganizmov, Bmo, Tvrdého č. 14 pod uvedenými číslami.The aforementioned drawbacks are largely eliminated by the process for preparing the nitrogen binder composition of the present invention, which is based on the use of some dearly in rhodorotropic bacteria which are capable of binding nitrogen from the air by expanding them during soil propagation, during their lifetime. synthesized forms of nitrogen into the soil such as nitrates, nitrites, ammonium, amino acids, small molecule proteins that bind to the organic matter of the soil from where their plant roots absorb. The continuously produced nitrogenous substances are not washed out of the soil and do not pollute the environment. The big advantage is that during the vegetative period nitrogen is supplied to plants continuously and harmoniously. Suitable species are the bacterial strain Azotobacter croococcum CCM 4642 and the bacterial strain Azospirillium brasiliense CCM 4644. These bacterial strains were deposited in the authenticated Czech Collection of Microorganisms, Bmo, Tvrdý no. 14 under the numbers indicated.
Bakteriálny kmeň Azotobacter croococcum sa najlepšie množí pri teplote od 16 °C do 30 °C, kým bakteriálny kmeň Azospirillium brasiliense má teplotné optimum od 24 “C do 38 °C. Týmito dvoma druhmi kmeňov baktérií sa naočkuje pôda a potom sa kultivujú v pôde zohriatej v jarnom období na 16 °C a v lete pri teplote pôdy 38 °C. Týmto spôsobom sa zabezpečuje rastlinám harmonické zásobovanie dusíkom.The bacterial strain Azotobacter croococcum is best propagated at a temperature of 16 ° C to 30 ° C, while the bacterial strain Azospirillium brasiliense has a temperature optimum of 24 ° C to 38 ° C. The two strains of bacteria are inoculated and then cultivated in soil heated to 16 ° C in the spring and 38 ° C in the summer. In this way, a harmonic nitrogen supply is provided to the plants.
Ďalej sa v pôde v dôsledku nadmerného používania umelých hnojív s obsahom fosforu nazhromaždilo veľmi veľa zlúčenín fosforu, ktorý je vo vode nerozpustný, a preto pre rastliny vyžadujúce fosfor je nevyužiteľný. Mikroorganizmus druhu Bacillus megatherium CCM 4643, ktorý bol uložený v autentizovanej Českej zbierke mikroorganizmov, Bmo, Tvrdého č. 14 pod uvedeným číslom, je schopný, v rámci svojho energetického metabolizmu meniť zlúčeniny fosforu s veľkými molekulami, ktoré sú vo vode nerozpustné na zlúčeniny fosforu vo vode rozpustné, a tým zásobovať pôdu, pre rastliny využiteľnými, rozpustnými zlúčeninami fosforu.Furthermore, very many phosphorus compounds, which are insoluble in water, have accumulated in the soil due to the excessive use of phosphorus-containing fertilizers and are therefore unusable for plants requiring phosphorus. The microorganism of the species Bacillus megatherium CCM 4643, which was deposited in the authenticated Czech collection of microorganisms, Bmo, Tvrdý no. 14 is capable of converting phosphorus compounds with large molecules which are water-insoluble into water-soluble phosphorus compounds as part of its energy metabolism and thereby supply the soil with plant-soluble, soluble phosphorus compounds.
Naočkovaním pôdy kmeňmi mikroorganizmov viažucich dusík a súčasným naočkovaním pôdy kmeňmi mikroorganizmov rozkladajúcich nerozpustné zlúčeniny fosforu vzniká takzvaná metabióza, kde viazače dusíka dodávajú potrebný dusík a mikroorganizmus Bacillus megatherium premieňa nerozpustné zlúčeniny fosforu na zlúčeniny fosforu vo vode rozpustné, pričom obidva druhy baktérií získavajú energiu, potrebnú na vlastný rast a množenie a zároveň dodávajú dusík a fosfor rastlinám.Inoculation of the soil with nitrogen-binding microorganisms and simultaneous inoculation of the soil with microorganisms decomposing insoluble phosphorus compounds results in so-called metabiosis, where the nitrogen binders supply the necessary nitrogen and the Bacillus megatherium converts the insoluble phosphorus compounds into the water-soluble phosphorus species. their own growth and multiplication, while supplying nitrogen and phosphorus to plants.
Pri výrobe jedlých olejov vzniká najmä na konci výroby olejový kal, ktorý sa už nedá ďalej čistiť, nie je vhodný na použitie v potravinárskom priemysle ani na iné využitie napr. v automobilovom priemysle. Likvidácia tohto olejového kalu nemôže prebiehať voľne v životnom prostredí, preto sú na jeho likvidáciu potrebné neutralizačné a čistiace zariadenia. Preto je výhodné využívať opísanú vlastnosť mikroorganizmov kmeňa baktérie Pseudomonas putida CCM 4641, ktorý bol uložený v autentizovanej Českej zbierke mikroorganizmov, Bmo, Tvrdého č. 14 pod uvedeným číslom. Tieto mikroorganizmy svojím pôsobením rozkladajú olejové zlúčeniny s otvoreným uhľovodíkovým reťazcom na glycerín a organické kyseliny.In the production of edible oils, especially at the end of production, oil sludge is formed which can no longer be cleaned; it is not suitable for use in the food industry or for other uses, e.g. in the automotive industry. The disposal of this oil sludge cannot take place freely in the environment, therefore neutralization and cleaning equipment are required for its disposal. Therefore, it is advantageous to utilize the described property of microorganisms of the strain Pseudomonas putida CCM 4641, which has been deposited in the authenticated Czech Collection of Microorganisms, Bmo, Tvrdý no. 14 under that number. These microorganisms, by their action, decompose open-chain oil compounds into glycerin and organic acids.
Olejový kal sa zmieša s úrodnou pôdou a táto zmes sa naočkuje kultúrami Azotobacter croococcum, Azospirillium brasiliense, Bacillus megatherium a potom aj kultúrou Pseudomonas putida, zmes sa niekoľkokrát premieša, obsah olejového kalu sa za dva-tri mesiace mineralizuje a rozkladá. Po aplikácii na omú pôdu spolu s veľkým množstvom mikróbov predstavuje výživnú látku s charakteristickými vlastnosťami maštaľného hnoja.The oil sludge is mixed with the fertile soil and inoculated with cultures of Azotobacter croococcum, Azospirillium brasiliense, Bacillus megatherium and then with Pseudomonas putida, mixed several times, and the oil sludge content is mineralized and decomposed in two to three months. When applied to my soil together with a large number of microbes, it is a nutrient with the characteristics of livestock manure.
Vynález je možné využiť tak, že hustá biomasa mikroorganizmov Azospirillium brasiliense, Azotobacter croococcum a Bacillus megatherium sa po nariedení na vhodnú koncentráciu v množstve desať litrov na hektár rozprašuje na pôdu, potom sa pôda prevracia do hĺbky sejby, čím sa pôda zároveň naočkuje tak, že sa môže čiastočne alebo iThe invention can be utilized by spraying the dense biomass of the microorganisms Azospirillium brasiliense, Azotobacter croococcum and Bacillus megatherium after dilution to a suitable concentration of 10 liters per hectare onto the soil, then turning the soil to the depth of sowing, thereby inoculating the soil. may partially or i
SK 279941 Β6 úplne upustiť od použitia umelých hnojív s obsahom dusíka a fosforu.276 completely abandon the use of fertilizers containing nitrogen and phosphorus.
Ďalej sa môže k uvedeným trom kultúram mikroorganizmov pridať ešte ďalšia kultúra mikroorganizmu Pseudomonas putida, a zmes sa používa tak, že na 10 ton pôdy sa pridá 10 litrov vhodne nariedenej zmesi mikroorganizmov a pôda sa viackrát premieša. Touto metódou sa môže rozložiť 500 litrov olejového kalu. Výsledným produktom spôsobu podľa vynálezu je výživná zmes bohatá na mikroorganizmy viažuce dusík, rozkladajúce fosfor a olejový sediment. Tento štvorkomponentový prípravok sa môže použiť ako náhrada za umelé hnojivo.Further, a further Pseudomonas putida culture can be added to the three microorganism cultures, and the mixture is used by adding 10 liters of a suitably diluted microorganism mixture per 10 tonnes of soil and mixing the soil several times. 500 liters of oil sludge can be decomposed by this method. The resulting product of the process according to the invention is a nutrient mixture rich in nitrogen-binding microorganisms, decomposing phosphorus and oil sediment. This four-component preparation can be used as a substitute for fertilizer.
Podstatou vynálezu je spôsob výroby zmesi mikroorganizmov na viazanie dusíka zo vzduchu, zvyšovanie rozpustnosti nerospustného fosforu v pôde a mineralizáciu kalu z jedlého oleja.The present invention provides a process for producing a mixture of microorganisms for binding nitrogen from air, increasing the solubility of non-permeable phosphorus in soil, and mineralizing sludge from edible oil.
Podľa vynálezu sa bakteriálny kmeň Azotobacter croococcum CCM 4642 množí na živnej pôde, ktorá neobsahuje organický ani anorganický dusík, submerznou fermentáciou pri teplote okolo 28 °C počas 22 hodín, potom sa ferment Azobacteru naočkuje 10 % -mi 22 hodinového inokula kmeňa baktérie Pseudomonas putida CCM 4641. Mikroorganizmus rada Azobacter počas 22 hodín naprodukuje do fermentačného média toľko dusíka koľko potrebuje mikroorganizmus rodu Pseudomas k svojmu rastu. V dvojitej fennentácii sa pokračuje ešte 22 - 24 hodín, pri prevzdušňovaní 0,6 litrov vzduchu na liter živnej pôdy, pri počte otáčok 120/min. a pri teplote 28 °C. Po ukončení kultivácie, po uplynutí 44 - 46 hodín, je pH kultivačnej pôdy v rozmedzí 7,5 - 8,5 a nárast 4 - ómliárd buniek/ml. média kmeňa Azotobacter croococcum a 150 - 200 miliónov bunick/ml. kmeňa Pseudomonasputida.According to the invention, the bacterial strain Azotobacter croococcum CCM 4642 is propagated on a nutrient-free medium containing no organic or inorganic nitrogen by submerged fermentation at about 28 ° C for 22 hours, then the Azobacter ferment is inoculated with a 10% 22 hour inoculum of Pseudomonas putida CCM 4641. A microorganism of the Azobacter family produces within 22 hours as much nitrogen as the microorganism of the genus Pseudomas needs to grow in the fermentation medium. The double fennentation is continued for 22-24 hours, with aeration of 0.6 liters of air per liter of nutrient medium, at a speed of 120 rpm. and at 28 ° C. At the end of the cultivation, after 44-46 hours, the pH of the culture medium is in the range of 7.5-8.5 and the growth of 4-cellliells / ml. medium of Azotobacter croococcum and 150-200 million cells / ml. strain of Pseudomonasputida.
Ďalej podľa vynálezu sa bakteriálny kmeň Azospirillium brasiliense CCM 4644 množí na živnej pôde, ktorá neobsahuje organický ani anorganický zdroj dusíka fermentáciou pri teplote 28 °C počas 22 hodín, potom sa pomnožená kultúra mikroorganizmu Azospirillium brasilierrse naočkuje 10 % -mi 22 hodinového inokula kmeňa baktérií Bacillus megatherium CCM 4643, pričom bakteriálny kmeň Azospirillium brasiliense do toho času naprodukuje do živného média toľko dusíka, koľko potrebuje mikroorganizmus Bacillus megatherium na svoj rast. V dvojitej submeiznej fermentácií sa pokračuje ešte 22 - 24 hodín pri prevzdušňovaní 1 liter vzduchu na 1 liter fermentačného média pri počte otáčok 120/minútu a pri teplote 28 °C. Po ukončení fermentácie je pH fermentačného média 6,8 - 8,5 a nárast kmeňa Azospirillium brasiliense 4-6 miliárd buniek/ml média a kmeňa Bacillus megatherium 200 - 300 miliónov buniek/ml média.Further, according to the invention, the bacterial strain Azospirillium brasiliense CCM 4644 is propagated on a culture medium which does not contain an organic or inorganic nitrogen source by fermentation at 28 ° C for 22 hours, after which a multiplied culture of Azospirillium brasilierrse is inoculated with 10% Bacillus strain. megatherium CCM 4643, whereby the bacterial strain Azospirillium brasiliense has so far produced as much nitrogen into the nutrient medium as the microorganism Bacillus megatherium needs to grow. Double sub-fermentation is continued for 22-24 hours while aerating 1 liter of air per liter of fermentation medium at 120 rpm and at 28 ° C. After fermentation, the pH of the fermentation medium is 6.8-8.5 and the growth of the Azospirillium brasiliense strain is 4-6 billion cells / ml medium and the Bacillus megatherium strain 200-300 million cells / ml medium.
Pri technickej realizácii vynálezu nevzniká žiadny vedľajší produkt. Biomasa mikroorganizmov sa môže využívať ako ferment, ktorý sa už ďalej nijako nespracováva. Voda z umývania fermentorov sa môže dostať do kanalizácie, pretože výhodne obohacuje vodu riek vzácnymi druhmi baktérií.In the technical implementation of the invention, no by-product is formed. Biomass of microorganisms can be used as a ferment that is no longer processed. The water from the washing of the fermenters can reach the sewage system as it advantageously enriches the water of the rivers with rare species of bacteria.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1Example 1
Do banky sa dá 200 ml zmesi zloženia dihydrogenfosforečnan draselný 0,3 g, chlorid vápenatý 0,2 g, síran horečnatý 0,3 g, síran draselný 0,2 g, chlorid sodný 0,4 g, chlorid železitý 0,05 g, uhličitan vápenatý 5,0 g, melasa 30,0 g, skvasiteľný cukor 10,0 g na 1000 ml živnej pôdy a počas 40 minút sa pôda sterilizuje pri teplote 125 °C, potom sa pridá 1 ml roztoku stopových prvkov zloženia kyselina bóritá 5,0 g, molybdénan amónny 6,0 g, jodid draselný 0,5 g, bromid sodný 0,5 g, síran zinočnatý 0,3 g, síran hlinitý 0,3 g na 1000 ml živnej pôdy. Do obsahu lyofylizovanej ampulky oboch bakteriálnych kmeňov Azotobacter croococcum a Azospirillium brasiliense sa pridá 5 ml sterilizovanej vody a vytvorí sa suspenzia, 1,0 ml -om suspenzie sa naočkujú obidve banky. Naočkované banky sa položia na horizontálne trepacie zariadenie a kultivujú sa pri teplote 28 °C a pri počte otáčok 160 za minútu počas 48 - 72 hodín, do vytvorenia dostatočného nárastu biomasy. Takto sa pripraví základné inokulum.200 ml of a mixture of potassium dihydrogen phosphate 0.3 g, calcium chloride 0.2 g, magnesium sulfate 0.3 g, potassium sulfate 0.2 g, sodium chloride 0.4 g, ferric chloride 0.05 g, calcium carbonate 5.0 g, molasses 30.0 g, fermentable sugar 10.0 g per 1000 ml of nutrient broth and sterilized at 125 ° C for 40 minutes, then 1 ml of the boric acid 5 trace element solution is added, 0 g, ammonium molybdate 6.0 g, potassium iodide 0.5 g, sodium bromide 0.5 g, zinc sulfate 0.3 g, aluminum sulfate 0.3 g per 1000 ml of culture medium. To the contents of the lyophilized ampoule of both the bacterial strains of Azotobacter croococcum and Azospirillium brasiliense, 5 ml of sterilized water is added and a suspension is formed, and 1.0 ml of suspension is inoculated with both flasks. The inoculated flasks are placed on a horizontal shaker and cultured at 28 ° C and 160 rpm for 48-72 hours, until sufficient biomass growth occurs. A basic inoculum is thus prepared.
Ďalej 400 ml živnej pôdy uvedeného zloženie sa dá do banky a naočkuje sa 3 - 4 ml -mi základného inokula a kultivuje sa na horizontálnom trepacom zariadení pri teplote 28 °C počas 48 hodín. Takto sa získa prípravné inokulum.Next, 400 ml of the nutrient broth of the above composition are placed in a flask and inoculated with 3-4 ml of basic inoculum and cultured on a horizontal shaker at 28 ° C for 48 hours. A preparative inoculum is thus obtained.
Ďalej 1000 litrov živnej pôdy sa sterilizuje vo fermentore pri 125 °C počas 40 minút, po ochladení sa naočkuje 1,6 1 -mi prípravného inokula. Naočkovaná pôda sa fermentuje počas 22 hodín pri teplote 28 °C za prevzdušňovania 0,6 litra vzduchu na 1 liter fermentačného média, pri počte otáčok 120 za minútu. Takto sa získa z oboch druhov mikroorganizmov prevádzkové inokulum vhodnej konzistencie. Na zamedzenie tvorby peny sa pridá odpeňovač v množstve 0,2 - 1,0 litrov/m3. Hodnota pH živnej pôdy je v rozsahu 6,8 - 7,0.Next, 1000 liters of nutrient broth are sterilized in a fermenter at 125 ° C for 40 minutes, and after cooling, they are inoculated with 1.6 l of preparative inoculum. The inoculated soil is fermented for 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of fermentation medium at 120 rpm. In this way, an inoculum of suitable consistency is obtained from both microorganisms. To prevent the formation of foam, an antifoam is added in an amount of 0.2 - 1.0 liters / m 3 . The pH of the broth is in the range 6.8 - 7.0.
Príklad 2Example 2
Do banky sa dá 200 ml živnej pôdy zloženia dihydrogenfosforečnan draselný 0,3 g, chlorid vápenatý 0,2 g, síran horečnatý 0,3 g, melasa 30,0 g, coom - steep 0,5 g, peptón 0,3 g, uhličitan vápenatý 5,0 g a počas 40 minút sterilizujeme pri 125 °C.200 ml of potassium dihydrogen phosphate 0.3 g, calcium chloride 0.2 g, magnesium sulphate 0.3 g, molasses 30.0 g, co-steep 0.5 g, peptone 0.3 g, sterilize calcium carbonate 5.0 g for 40 minutes at 125 ° C.
Do obsahu lyofylizovanej ampulky oboch bakteriálnych kmeňov Bacillus megatherium a Pseudomonas putida sa pridá 5 ml sterilizovanej vody a vytvorí sa suspenzia, potom 1,0 ml -om suspenzie sa naočkujú obidve banky. Naočkované banky sa položia na horizontálne trepacie zariadenie a kultivujú sa pri teplote 28 °C počas 48 hodín pri počte otáčok 160 za minútu.To the contents of the lyophilized ampoule of both bacterial strains of Bacillus megatherium and Pseudomonas putida is added 5 ml of sterilized water and a suspension is formed, followed by inoculation of the two flasks with 1.0 ml of suspension. The inoculated flasks are placed on a horizontal shaker and cultured at 28 ° C for 48 hours at 160 rpm.
Ďalej 400 ml uvedenej živnej pôdy sa dá do banky a naočkuje sa 1 - 2 ml -mi základného inokula a mieša sa na horizontálnom trepacom zariadení pri teplote 28 °C počas 24 - 48 hodín, pričom mikroskopickým sledovaním sa zisťuje vegetatívny stav kultúry a vznik spór. Týmto postupom sa získa prípravné inokulum.A further 400 ml of said culture medium is placed in a flask and inoculated with 1-2 ml of basic inoculum and mixed on a horizontal shaker at 28 ° C for 24-48 hours, the microscopic observation of the vegetative state of the culture and the formation of spores. . This procedure yields a preparative inoculum.
Ďalej 1000 litrov živnej pôdy sa sterilizuje vo fermentore pri 125 °C počas 40 minút, po ochladení sa naočkuje 3 kusmi prípravného inokula s obsahom 400 ml. Naočkovaná pôda sa fermentuje počas 22 hodín pri teplote 28 °C za prevzdušňovania 0,6 litrov vzduchu na 1 liter živnej pôdy, pri počte otáčok 120 za minútu. Na zamedzenie tvorby peny sa pridá odpeňovač v množstve 0,2 - 1,0 litrov/m3. Mikroskopicky sa sleduje nárast kultúry tak, aby prebiehal len do vegetatívneho stavu bez tvorby spór. Hodnota pH živnej pôdy je v rozsahu 6,8 - 7,0.Next, 1000 liters of nutrient broth are sterilized in a fermenter at 125 ° C for 40 minutes, inoculated with 3 pieces of 400 ml preparative inoculum after cooling. The inoculated soil is fermented for 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of nutrient medium at 120 rpm. To prevent the formation of foam, an antifoam is added in an amount of 0.2 - 1.0 liters / m 3 . The growth of the culture is monitored microscopically so that it proceeds only to the vegetative state without the formation of spores. The pH of the broth is in the range 6.8 - 7.0.
Príklad 3Example 3
V dvoch fermentoroch s celkovým obsahom 10-12 m3, jednotlivého užitočného objemu 5,0 - 6,0 m3, sa sterilizuje 4,5 a 4,5 m3 živnej pôdy bez obsahu dusíka, opísanej v príklade 1 a naočkuje sa 10 % -mi prevádzkového inokula s obsahom bakteriálneho kmeňa Azotobacter croococcum podľa príkladu 2. Potom sa kultivuje pri teplote 28 °C počas 22 hodín za súčasného prevzdušňovania 0,6 litrov vzduchu na liter živnej pôdy, pri počte otáčok 120 za minútu. Potom sa fermentovaná pôda naočkuje ešte 10 % - mi kultúry mikroorganizmu Pseudomonas putida podľa príkladu 2, a ďalej sa pokračuje v dvojitej fermentácií ešte 22 - 24 hodín. Po ukončení 44 - 46 hodinovej fermentácie je pH živnej pôdy 7,7 - 8,5 a bakteriálny kmeň Azotobacter croococcum dosiahol nárast 4-8 miliárd buniek /ml média a kmeň Pseudomonas putida 200 - 300 miliónov buniek/ml média.In two fermenters with a total content of 10-12 m 3 , a single useful volume of 5.0 - 6.0 m 3 , 4.5 and 4.5 m 3 of nitrogen-free broth described in Example 1 are sterilized and inoculated with 10 % of the inoculum containing the bacterial strain Azotobacter croococcum according to Example 2. It is then cultured at 28 ° C for 22 hours while aerating 0.6 liters of air per liter of culture medium, at a speed of 120 rpm. The fermented broth is then inoculated with a 10% culture of Pseudomonas putida according to Example 2, and the double fermentation is continued for 22-24 hours. After 44-46 hours of fermentation, the pH of the broth is 7.7-8.5 and the bacterial strain Azotobacter croococcum achieved an increase of 4-8 billion cells / ml medium and the Pseudomonas putida strain 200-300 million cells / ml medium.
Do dvoch fermentorov s užitočným objemom 5,5 - 6,0 m3 sa vloží 4,5 a 4,5 m3 živnej pôdy bez dusíka opísanej v príklade 1 a naočkuje sa 10 % -mi prevádzkového inokula s obsahom bakteriálneho kmeňa Azospirillium brasitiense podľa príkladu 1, kultivácia trvá 22 hodín pri teplote 28 °C pri prevzdušňovaní 0,6 litrov vzduchu na liter živnej pôdy, pri počte otáčok 120 za minútu. Fermentačné médium sa potom naočkuje ešte 10 % - mi prevádzkového inokula kultúry mikroorganizmu Bacillus megatherium podľa príkladu 1, a ďalej sa pokračuje v dvojitej fennentácii ešte 22 - 24 hodín. Po ukončení lamentácie je pH fermentačného média 7,5 - 8,5 a kmeň Azospirillium brasiliense dosiahol nárast 4 - 6 miliárd buniek /ml média a kmeň Bacillus megatherium 200 - 300 miliónov buniek/ml média.4.5 and 4.5 m 3 of nitrogen-free broth described in Example 1 are introduced into two fermenters with a useful volume of 5.5-6.0 m 3 and inoculated with a 10% operational inoculum containing the bacterial strain Azospirillium brasitiense according to of Example 1, the cultivation lasted 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of nutrient medium at 120 rpm. The fermentation medium is then inoculated with a 10% operational inoculum of a Bacillus megatherium culture according to Example 1, and the double fermentation is continued for 22-24 hours. At the end of the lamentation, the pH of the fermentation medium is 7.5-8.5 and the Azospirillium brasiliense strain achieved an increase of 4-6 billion cells / ml medium and the Bacillus megatherium strain 200-300 million cells / ml medium.
Ďalej zmiešaním oboch typov fermentov a ich homogenizovaním sa získa štvorzložkový výsledný produkt zloženia: Azotobacter croococcum 2-4 miliárd buniek/ ml, Azospirillium brasilierrse 2-4 miliárd buniek/ml, Pseudomonas putida 150 - 200 miliónov buniek/ml aBacillus megatherium 150 - 200 miliónov buniek/ml, pričom v množenej biomase je celkom 4,3 miliárd buniek/ml až 8,4 miliárd buniek/ml živného média a pH je v rozmedzí 7,5 - 8,5.Further mixing and homogenizing both types of ferments yields a four-component final product of the composition: Azotobacter croococcum 2-4 billion cells / ml, Azospirillium brasilierrse 2-4 billion cells / ml, Pseudomonas putida 150-200 million cells / ml and Bacillus megatherium 150-200 million cells / ml, wherein the multiplied biomass has a total of 4.3 billion cells / ml to 8.4 billion cells / ml of nutrient medium and the pH is in the range of 7.5-8.5.
Priemyselná využiteľnosťIndustrial usability
Opísaným spôsobom podľa vynálezu sa pripraví zmes mikroorgamizmov na viazanie dusíka zo vzduchu, na zvyšovanie rozpustnosti zlúčenín fosforu vo vode a na rozklad potravinárskeho olejového sedimentu, ktorá má využitie v poľnohospodárstve.According to the method of the invention, a mixture of microorganisms is prepared to bind nitrogen from air, to increase the water solubility of phosphorus compounds, and to decompose a food oil sediment for use in agriculture.
Claims (1)
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SK250-97A SK279941B6 (en) | 1997-02-25 | 1997-02-25 | Process for preparing a mixture of microorganisms binding nitrogen from the air, increasing phosphorous compounds solubility and decomposing oil sediments |
| AU61296/98A AU6129698A (en) | 1997-02-25 | 1998-02-19 | Process for extracting nitrogen from air, phosphorus compounds from the soil andfor decomposing oil sediments |
| PCT/SK1998/000003 WO1998037038A2 (en) | 1997-02-25 | 1998-02-19 | Process for extracting nitrogen from air, phosphorus compounds from the soil and for decomposing oil sediments |
| HU0002253A HU225130B1 (en) | 1997-02-25 | 1998-02-19 | Microbal mixture for binding nitrogen from air, phosphorus compounds from the soil and for decomposing oil sediments and process for preparing thereof |
| CZ0302599A CZ299601B6 (en) | 1997-02-25 | 1998-02-19 | Process for preparing mixture of microorganisms for binding nitrogen from air for increasing solubility of phosphorus compounds and for decomposition of foodstuff oil sediment |
| DE19880157T DE19880157B4 (en) | 1997-02-25 | 1998-02-19 | A method of preparing a microorganism mixture for binding atmospheric nitrogen, increasing the solubility of phosphorus compounds and decomposing food oil sediment and said mixture |
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| Application Number | Priority Date | Filing Date | Title |
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| SK250-97A SK279941B6 (en) | 1997-02-25 | 1997-02-25 | Process for preparing a mixture of microorganisms binding nitrogen from the air, increasing phosphorous compounds solubility and decomposing oil sediments |
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| SK25097A3 SK25097A3 (en) | 1998-08-05 |
| SK279941B6 true SK279941B6 (en) | 1999-06-11 |
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| AU (1) | AU6129698A (en) |
| CZ (1) | CZ299601B6 (en) |
| DE (1) | DE19880157B4 (en) |
| HU (1) | HU225130B1 (en) |
| SK (1) | SK279941B6 (en) |
| WO (1) | WO1998037038A2 (en) |
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| HUP0000207A2 (en) * | 2000-01-21 | 2001-12-28 | Bio-Mikro Kft. | Composition and method for treating sclerotinia infection |
| SK3862000A3 (en) * | 2000-03-16 | 2001-10-08 | Arpad Pollak | Method for the preparation of microorganism mixture for binding air nitrogen and for increasing the phosphorous compound solubility in soil and mixture listed above |
| HU230555B1 (en) | 2001-08-13 | 2016-12-28 | Biofil Kft. | Environment-friend micro-organism produce and producing thereof |
| CN101497542B (en) * | 2008-11-19 | 2011-07-06 | 黄晓东 | Biological preparation containing plant growth promoting bacteria, as well as preparation and use thereof |
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| US4952229A (en) * | 1983-09-27 | 1990-08-28 | Hugh M. Muir | Plant supplement and method for increasing plant productivity and quality |
| CZ283310B6 (en) * | 1991-01-31 | 1998-02-18 | Vysoká Škola Chemicko-Technologická | Method of biological maintenance of oil-contaminated soil by employing selected micro-organisms |
| HU207751B (en) * | 1991-02-08 | 1993-05-28 | Phylaxia Oltoanyagtermeloe | Process for producing composition for utilizing nitrogen of the air and phosphorous of the soil for plant |
| CZ280509B6 (en) * | 1994-03-31 | 1996-02-14 | Vysoká Škola Chemicko-Technologická | Disposal of used cutting and hydraulic liquids and oils |
| RU2072756C1 (en) * | 1994-06-15 | 1997-02-10 | Научно-исследовательский и проектно-конструкторский институт охраны окружающей среды в угольной промышленности | Method for recultivation of disturbed land after coal mining |
| ES2093559B1 (en) * | 1995-05-04 | 1997-07-01 | Consejo Superior Investigacion | BACTERIAL FERTILIZER AND PROCEDURE FOR OBTAINING. |
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| Publication number | Publication date |
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| AU6129698A (en) | 1998-09-09 |
| WO1998037038A3 (en) | 1998-11-12 |
| CZ299601B6 (en) | 2008-09-17 |
| HU225130B1 (en) | 2006-06-28 |
| DE19880157D2 (en) | 2000-03-16 |
| CZ302599A3 (en) | 2000-01-12 |
| WO1998037038A2 (en) | 1998-08-27 |
| HUP0002253A2 (en) | 2000-11-28 |
| SK25097A3 (en) | 1998-08-05 |
| HUP0002253A3 (en) | 2001-12-28 |
| DE19880157B4 (en) | 2013-04-11 |
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