WO2007032704A1 - Procede de production d'une preparation biologique rhoder destinee a eliminer le petrole et les produits petroliers d'eau, de sols et de boues d'hydrocarbures residuaires - Google Patents

Procede de production d'une preparation biologique rhoder destinee a eliminer le petrole et les produits petroliers d'eau, de sols et de boues d'hydrocarbures residuaires Download PDF

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Publication number
WO2007032704A1
WO2007032704A1 PCT/RU2006/000403 RU2006000403W WO2007032704A1 WO 2007032704 A1 WO2007032704 A1 WO 2007032704A1 RU 2006000403 W RU2006000403 W RU 2006000403W WO 2007032704 A1 WO2007032704 A1 WO 2007032704A1
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Prior art keywords
oil
cultivation
strains
bkm
preparation
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PCT/RU2006/000403
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English (en)
Russian (ru)
Inventor
Valentina Pavlovna Murygina
Sergey Vladimirovich Kalyuzhnyi
Natalia Evgenievna Voishvillo
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Valentina Pavlovna Murygina
Kalyuzhnyi Sergey Vladimirovic
Natalia Evgenievna Voishvillo
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Publication of WO2007032704A1 publication Critical patent/WO2007032704A1/fr

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to biotechnology and ecology. It can be used in the microbiological industry and is intended to produce a bacterial oil destructor and protect the environment (soil, soil, oil sludge, fresh and mineralized waters) from hydrocarbon contamination of oil and oil products.
  • the composition of the drug Devoroil includes strains of Pseudomopas stutzeri, Rhodosossus eruthoris, Rhodosossus maris, Rhodosossus sp., Yarrowi lirolutisa, (previously Sapdida sp.) [5,
  • Devoroil is also obtained by deep cultivation of the microorganisms included in its composition in the nutrient medium and subsequent drying of the resulting biomass.
  • Devoroil consists of microorganisms of various taxonomic and species belonging, requiring separate cultivation, which significantly complicates and increases the cost of the drug production process.
  • the preparation [9] which consists of two mesophilic pseudomonads, micrococcus and two psychrophilic pseudomonads and xanthomonos, all six strains, which, when receiving the seed, are grown separately and cultivated in a fermenter at 2O 0 C with the addition of crude oil as an inductor and then grown for 120 hours at a temperature of 3 0 C on peat with the addition of carbamide, superphosphate and potassium sulfate.
  • the low activity of the drug consisting of heterogeneous microorganisms - bacteria and yeast Acipetobaster olevorum, Musosossus lactis, Sapdida trisalis, Sapdida guilliermopdii, obtained by co-cultivation [10], was established
  • More preferable for obtaining biologics with a high titer of viable cells, in the case of applying the stage of co-cultivation, are associations of microorganisms of the same taxonomic affiliation.
  • this method in the first stage of preparation of the drug, deep cultivation is carried out on a liquid nutrient medium, in the second stage, bacteria are co-grown on the surface of a solid carrier, which is sterile peat, i.e. surface cultivation is carried out.
  • Preliminary deep cultivation is carried out either in 5 l large bottles or in a 100 l fermenter.
  • sterile plastic bags are prepared, which are filled with sterile peat in an amount of 0.6 kg and sown with 150 ml of seed obtained at the stage of deep cultivation.
  • the packages are kept hermetically sealed for 7 days. at 30 0 C and receive a drug with a titer of viable cells 10 12 per gram.
  • the disadvantage of this method is: the complexity of the stage of surface cultivation, namely: the preparation of sterile plastic bags, packaging sterile peat in sterile bags, its inoculation with bacteria and the duration of their cultivation.
  • the method is difficult to scale due to its low manufacturability.
  • a preparation is obtained that consists not of free, but of immobilized cells, and there are no examples in the description confirming their hydrocarbon-oxidizing activity, and there are no conditions and storage periods of the resulting preparation.
  • the disadvantage of this method is the need for a sterile artificial carrier and long-term surface cultivation of cells on a carrier, which characterizes
  • the above methods obtain oil-degrading preparations consisting of immobilized cells, the advantage of which is that they are less sensitive to the effects of adverse conditions.
  • the disadvantage of immobilized cells is that access to hydrophobic substrates, in particular oil and products of its processing, is difficult for such cells.
  • the hydrocarbon-oxidizing activity of biological products intended for cleaning environmental objects from oil and oil products is determined not only by their composition, but also largely depends on the commodity form in which they are offered to the consumer.
  • Known biological products are obtained in the form of suspensions, emulsions, pastes, powders [2-11, 13, 14].
  • each form has its own advantages and disadvantages.
  • the number of living microorganisms is usually lower than in suspension or paste [15], since in domestic factories for obtaining dry forms, primarily non-medical preparations, spray drying is generally practiced, which is considered less energy-intensive and cheaper than lyophilization , however, the quality of biological products is significantly deteriorating.
  • dry preparations have an undeniable advantage: they are more convenient for transportation to their places of use and have a longer shelf life.
  • the authors of dry forms of biological products recommend activation of the microorganisms included in their composition before use [4, 16].
  • Activation of microorganisms in a dry preparation involves the preparation of a liquid growth medium with nutrients, the introduction of a hydrocarbon as a carbon source, mixing and aeration for 12-24 hours [4, 16], which is not always possible to perform in the field.
  • Liquid and pasty preparations unlike dry forms, do not require activation, and they are used in contaminated places located near production, since the shelf life of such biological products at low temperatures [14], as a rule, does not exceed 2 months, and their transportation over long distances is not advisable.
  • SUBSTITUTE SHEET (RULE 26) 1.0, monosubstituted sodium phosphate -1.5, aqueous magnesium sulfate 0.2, aqueous manganese sulfate 0.02, to which sea salt 30-50 and oil 40 ml are added, pH 7.0-7.4 .
  • sea salt 30-50 and oil 40 ml are added, pH 7.0-7.4 .
  • subsequent seeding of strain cells on meat-peptone agar (MGLA) diluted 1: 1 with the mineral medium of the above composition to identify the R-forms of strains is mandatory. This approach allows you to select how to obtain the R-forms of rhodococci, and their adaptation to. increased concentration of petroleum products in the medium and to an increased content of mineral salts in the medium.
  • the selected R-forms of the strains included in the preparation are able to multiply and degrade oil hydrocarbons in a wide range of salinity (0.05-10%) (optimal salinity of the medium 3-5%) and temperatures (8-35 0 C) (optimal temperature 25-27 0 C).
  • this method of obtaining the drug was developed, in fact, for laboratory conditions, because the biomass of R-forms of rhodococci at the age of 7-10 days grown on mattresses on an agar medium is proposed to be used for the preparation of a biological product.
  • depleted media containing oil as the sole carbon source are used for cultivation, which makes the method not technologically advanced.
  • the R-forms of rhodococci that are part of the preparation are proposed to be grown in a liquid medium, which includes corn extract and microbial biomass fermentolizate (BVC) and provides separate culture of each strain.
  • BVC microbial biomass fermentolizate
  • the biological product is obtained by combining separately obtained biomass of each of the strains.
  • the phylogenetic position of the strains of Rhodococcus ruber BKM Ac-1513 D and Rhodosossus eruthoris BKM Ac-1514 D among representatives of the genus Rhodocossus was determined (Fig. 2).
  • Rhodosossus ruber BKM Ac-1513 D predominantly actively breaks down cyclic and polycyclic hydrocarbons
  • Rhodosossus eruthoris BKM Ac-1514 D breaks down primarily aliphatic hydrocarbons.
  • the technical result of the invention is to develop a technology that allows you to organize the process of scaled-up production of the bacterial preparation Roder with higher oil-oxidizing activity compared to the prototype [8], reduces the time of its production without compromising efficiency and quality characteristics. This is achieved by the fact that at the last stage of the preparation of Roder, when the biomass of the strains of the R-forms Rhodosossus eruthorisol BKM Ac-1514 D and Rhodosossus ruber BKM Ac-1513 D is increased, they are jointly cultured in a common fermenter on a selected nutrient medium, which, as the only carbon source contains glucose.
  • the process is conducted on a nutrient medium containing yeast autolysate or fish hydrolyzate as a source of nitrogen and other organic substances.
  • the biomass obtained in the fermenter is concentrated, stabilized and optionally dried.
  • Nutrient media contain organic sources of carbon, nitrogen, phosphorus, and medium II WG and III DAG also contain mineral elements.
  • Each strain of Rhodosossus ruber BKM Ac-1513 D and Rhodosossus eruthorosol BKM Ac-1514 D is cultivated individually in flasks with stirring and aeration on a rocking chair at a temperature not exceeding 29 C, because when growing biomass of strains at a temperature below 20 0 C or above 29 0 C, less active hydrocarbon oxidizing cells grow.
  • each strain individually, is introduced into the fermenter-inoculator with a medium of a composition different or the same as that used in the flasks, and cultivated with stirring and aeration at a temperature not lower than 20 0 C and not higher than 29 0 C.
  • Each strain is grown. individually in inoculant fermenters in accordance with their physiological characteristics.
  • the strain of Rhodosossus ruber BKM Ac-1513 D is grown for 30-36 hours, and the strain of Rhodosossus eruthorosol BKM Ac-1514 D is grown for 20-24 hours.
  • the inoculum from two fermenters is fed into one fermenter with the medium of the same composition, introducing strains in the specified ratio (1.5: 1) as seed. which allows you to reduce the period of co-culture to 26-28 hours and get cells of both strains with high hydrocarbon • oxidizing activity, and a culture fluid with a final
  • SUBSTITUTE SHEET (RULE 26) a neutral pH value, while the process is conducted in a specially worked out mode of oxygen supply and mixing.
  • the biomass obtained by co-culturing two strains of these rhodococci is concentrated with stabilizers and used as such or a dry form of the preparation is obtained under gentle conditions.
  • the conditions for obtaining a dry form of the drug were also worked out in accordance with the physiological characteristics of the strains.
  • the possibility of using Roder both in dry form and in the form of a concentrated suspension is its advantage, because most fully meets the requirements of consumers.
  • the joint cultivation of strains at the last stage allows us to simplify and reduce the cost of the process of scaled (industrial) production of the drug, to obtain the final product with a higher titer of hydrocarbon oxidizing cells and with a neutral pH of the culture fluid, to fully utilize (95-98%) the main components of the nutrient medium. This indicates the achievement of a technical result in the implementation of this method of industrial production of the drug Roder.
  • the preparation in the form of a concentrated suspension, obtained by the proposed method, at room temperature retains hydrocarbon oxidizing activity for 30 days, and at a storage temperature of +4 to +8 0 C for up to 2 months.
  • Roder controls the viability and hydrocarbon oxidizing activity of the strains and the drug.
  • the ready-made biological product Roder is either a pinkish-brownish concentrate of living cells of the strains mentioned above, with residues of the nutrient medium and protective composition, packaged in plastic cans from 1.0 l to 20 l, or a dry powder from light cream to pinkish brown colors with a specific odor with a mass fraction of moisture from 3% to 7%, packaged in packages from 0.5 kg to 25 kg, which allow preserving the activity of a biological product for up to 18 months at a temperature of +4 0 C to + 8 ° C. With the access of oxygen and higher humidity of the dry form of the drug, its shelf life decreases. Before use, the biological product in dry form or in the form of a concentrated suspension is sufficient to dilute with water.
  • the bacterial preparation obtained in this way / in the form of a concentrated suspension and in the form of a powder was tested in pilot and field conditions.
  • the fermentation time is reduced to 28 hours.
  • the culture fluid obtained by separate cultivation of the strains is combined (option 1) and concentrated by centrifugation as well as the culture fluid after co-cultivation (option 2).
  • the number of CFU / ml (colony of forming units) of heterotrophs (HT) and hydrocarbon-oxidizing bacteria (SVR), the weight of dry biomass and pH are determined (Table 3).
  • the titer of hydrocarbon oxidizing cells is 1-2 orders of magnitude higher than with separate.
  • the pH of the culture fluid is higher, and the density of the obtained biomass is 32-65% higher.
  • Example 2 Inoculum in flasks was prepared as described in example 1 and transferred to a 16 L fermenter with I-BVC medium (Table 2) each strain individually. Cultivation was carried out at a temperature of 28 0 C with aeration and stirring. R. erutorolis Ac-1514 D was cultured for 28 hours, and R. ruber Ac-1513 D for 40 hours. Seeds from the fermenters were combined into a 100-liter fermenter with I-BVA medium and cultured for 28 hours. Culture liquid concentrated by ultrafiltration, stabilized with polyglucin and lactose, packaged in cans. The SVR titer of the cells was 2.3 * 10 in 1 ml. The drug was used to clean a reservoir and marshy soil, accidentally contaminated with oil in Western Siberia (table. 4).
  • the roder biological product in the form of a concentrated suspension in 3 treatments reduced the concentration of hydrocarbons in water (pond) by 96%, and on marshy soil - by 94% in field trials in Western Siberia.
  • Example 3 Inoculum of both strains was grown in flasks on medium I-BVK. On a medium of the same composition, subsequent cultivation was carried out according to example 2. The titer of the finished product was 1.8 * 10 9 CFU / ml.
  • the drug was used in a pilot experiment on the soil (chronic oil pollution) in Urai (Western Siberia). The soil contaminated with oil with a hydrocarbon concentration (HC) of 178.2 g / kg of soil was cleaned using Putidul - dry powder, Devoroyl - dry powder, which was obtained by co-cultivation of microorganisms, its constituents and belonging to different genera, Nikoil - concentrated suspension; and Roder - concentrated suspension. Preparations were prepared for work as described in the instructions for each drug. The best results were obtained with Roder (Table 5).
  • SUBSTITUTE SHEET (RULE 26) Example 4.
  • the drug was prepared according to example 3, but used the medium P-RG.
  • the SVR titer of the cells was 3.3 * 10 12 in 1 ml.
  • the resulting preparation was used for bioremediation of sandy and peaty soil contaminated with accidentally spilled oil, the Republic of Komi, Timan-Pechora gas and oil province (Table 6). Weather conditions did not always favor the bioremediation process; nevertheless, as a result of 2-3 soil treatments with Roder biological product, hydrocarbon pollution was reduced by 21-75%.
  • Example 5 The drug was prepared in the form of a dry powder. Cultivation was carried out analogously to example 3, but medium III-DAG was used. The SVR titer of the cells was 3.0 * 10 and in 1 g of dry preparation. The resulting preparation was used to purify stormwater from oil products in the city of Stupino, Moscow Region, as well as for bioremediation of oil sludge, China, Shandong province (Table 7). Oil sludge was previously modified to create conditions favorable for the work of bacteria-destructors of the biological product Roder. The results are shown in table. 7 show that the Roder drug in dry form during one warm season in storm drains almost at a constant flow of oil products reduced the total concentration of hydrocarbons in water by 50-60%. For three treatments of oil sludge, Roder reduced the concentration of HC by 53-58%, while the alternative method of reclamation of oil sludge - composting - reduced the HC in oil sludge by only 31%.

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Abstract

La présente invention relève du génie biologique, et se rapporte à un procédé permettant de produire une préparation biologique Rhoder destinée à éliminer le pétrole et les produits pétroliers d'eau, de sols et de boues d'hydrocarbures résiduaires. Le procédé selon l'invention consiste : à préparer des clones de forme R de souches de Rhodococcus ruber BKM Ac-1513D et de Rhodococcus erythropolis BKM Ac-1514 D ; à sélectionner les clones les plus actifs ; à cultiver séparément des souches contenues dans la préparation biologique dans un milieu nutritif liquide de composition principalement minérale, en ajoutant de l'azote organique et du carbone ; à poursuivre ensuite la culture des souches dans un fermenteur commun, dans un milieu nutritif contenant du glucose comme source de carbone ; puis à concentrer et à stabiliser le liquide de culture, et à procéder éventuellement au séchage du produit obtenu.
PCT/RU2006/000403 2005-09-13 2006-07-28 Procede de production d'une preparation biologique rhoder destinee a eliminer le petrole et les produits petroliers d'eau, de sols et de boues d'hydrocarbures residuaires WO2007032704A1 (fr)

Applications Claiming Priority (2)

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RU2005128330 2005-09-13
RU2005128330/13A RU2295403C1 (ru) 2005-09-13 2005-09-13 Способ получения бактериального препарата родер для очистки почв, почвогрунтов, нефтешламов, пресных и минерализованных вод от нефти и нефтепродуктов

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RU2499636C1 (ru) * 2012-03-20 2013-11-27 Общество с ограниченной ответственностью "Уралэкоресурс" (ООО "Уралэкоресурс") Способ биоремедиации нефтезагрязненных почвогрунтов
RU2650864C1 (ru) * 2017-09-26 2018-04-17 Забокрицкий Александр Николаевич Биологический деструктор несимметричного диметилгидразина
PL236100B1 (pl) * 2018-01-19 2020-11-30 Univ Przyrodniczy W Poznaniu Konsorcjum bakteryjno-grzybowe i sposób bioremediacji gleby skażonej substancjami ropopochodnymi
CN111471612B (zh) * 2020-03-10 2022-09-30 中国水产科学研究院南海水产研究所深圳试验基地 一种净化海水池塘养殖尾水中无机氮磷的赤红球菌hdrr2y及其应用
CN111471611B (zh) * 2020-03-10 2022-09-27 中国水产科学研究院南海水产研究所深圳试验基地 一种净化海水池塘养殖尾水中无机氮磷的赤红球菌hdrr1及其应用

Citations (3)

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WO1995031408A1 (fr) * 1994-05-11 1995-11-23 Aktsionernoe Obschestvo Zakrytogo Tipa 'biotekhinvest' Preparation biologique et procede d'extraction de contaminants a base de produit de petrole brut et de petrole se trouvant dans de l'eau et de la terre
RU2138451C1 (ru) * 1997-12-05 1999-09-27 Саксон Валерий Михайлович Биопрепарат для очистки объектов окружающей среды от нефти и нефтепродуктов
RU2174496C2 (ru) * 1999-05-31 2001-10-10 Мурыгина Валентина Павловна Биопрепарат "родер" для очистки почв, почвогрунтов, пресных и минерализованных вод от нефти и нефтепродуктов

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031408A1 (fr) * 1994-05-11 1995-11-23 Aktsionernoe Obschestvo Zakrytogo Tipa 'biotekhinvest' Preparation biologique et procede d'extraction de contaminants a base de produit de petrole brut et de petrole se trouvant dans de l'eau et de la terre
RU2138451C1 (ru) * 1997-12-05 1999-09-27 Саксон Валерий Михайлович Биопрепарат для очистки объектов окружающей среды от нефти и нефтепродуктов
RU2174496C2 (ru) * 1999-05-31 2001-10-10 Мурыгина Валентина Павловна Биопрепарат "родер" для очистки почв, почвогрунтов, пресных и минерализованных вод от нефти и нефтепродуктов

Non-Patent Citations (2)

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Title
MURYGINA V.P. ET AL.: "Ochista vodnoi poverkhnosti i gruntov ot neftyanykh zagruzheny biopreparatom "Roder"", EKOLOGYA I PROMYSHLENNOST ROSSII, August 1999 (1999-08-01), pages 16 - 19 *
TASKAEV A.I. ET AL.: "Opyt biologicheskoi rekultivatsii zemel v uslovyakh krainego sereva", EKOLOGYA I PROMYSHLENNOST ROSSII, SPETSVYPUSK, 2004, pages 29 *

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