SU959634A3 - Process for producing fermentation broth - Google Patents

Abstract

1531970 Producing xanthomonas colloids PFIZER Inc 5 Aug 1977 [5 Aug 1976 3 May 1977] 33003/77 Heading C3U A fermentation medium containing Xanthomonas hydrocolloids is obtained by cultivating aerobically a Xanthomonas microorganism, preferably X. compestris in an aqueous nutrient containing a carbohydrate, a nitrogen source and 0À1-10 g/l of an assimilable Krebs cycle acid, 1-200 p.p.m. of chelated calcium and 0À25-20 p.p.m. of iron, and trace elements until at least 100 p.p.m. of colloid is present whereby the whole broth produces mobility control solutions suitable for subterranean oil recovery which are free of insoluble matter having particle sizes of above 3 microns.

Description

ethylene diamine tetraacetic acid is added to form chelate compounds. After cultivation, formaldehyde was introduced into the fermentation broth in an amount of 0.2-10 g / l. As the nitrogen source, ammonium, sodium and potassium nitrates are used. The water for the preparation of the nutrient medium contains less than 20 g / l of calcium and other precipitating cations in the form of phosphates. After cultivation, fermentation. The broth is aged for 3-4 days to obtain a broth that is practically free of insoluble particles greater than the size of 0.65 µm. The growth medium for this microorganism may be a UM broth (Difco) or a medium containing crude glucose (cerelose), sodium and potassium phosphates, magnesium sulfate and any organic compound that is a source of nitrogen, for example, soy bean enzyme or casein enzyme evaporation . After aerobic propagation for about 30 hours at 25 ° C, an aliquot of the broth is transferred to a fermenter to effect the second inoculation step. Preferred carbohydrates are glucose, maltose, fructose, filtered starch, or mixtures thereof. The preferred nitrogen source is inorganic nitrate: ammonium nitrate, sodium nitrate and potassium nitrate in predetermined concentrations. Magnesium, as one of the necessary microelements, is used in the form of MgSO-7HnO, or bitter salt, by adding it in a concentration of up to 1 g / l, along with traces of magnesium and iron. The chelating agent is ethylenediaminetetraacetic acid or, preferably, citric acid, which is used as a growth-promoting agent, as well as a calcium passivator / calcium carbonate. As a buffer for a medium providing a pH value of 8.5 to 8.5, preferably 6.0 to 7.5, large amounts of potassium mono- and diphosphates are added. After aerobic propagation for an hour at, preferably at 28-30 ° C, an aliquot of the solution is introduced into a fermentation tank containing the medium that provides the substance. This medium is similar in composition to the medium in the second stage of inoculation, except that instead of potassium phosphates, it is preferable to use sodium phosphates (due to their low cost) and a small amount of calcium in the form of salts, such as calcium chloride or nitrate. calcium, or oxides, which are added to increase xanthan yield. The amount of added calcium depends on the amount of calcium present in the water used in the preparation of the medium, the source of nitrogen used and the species and chains of the Xanthomonas microorganism used. If ammonium nitrate is used together with sodium nitrate or potassium nitrate, less calcium is needed (about 27 ppm) ). For the medium medium halfway, such deionized water can be used, distilled water - or 1 water containing less than 20 hours per million of calcium and other cations precipitated during the formation of phosphates. Calcium ions can be added in the required concentration. The role of calcium ions in ensuring the production of xanthate is great, but for the proposed method, the most important is to prevent excessive precipitation of calcium cations and other cations in the form of insoluble phosphate salts. This is done by adding a chelating reagent, such as ethylenediaminetetraacetic acid. An important parameter for ensuring the growth of Xanthomonas bacteria is the pH of the medium. Preferred values are from 6.0 to 7.5. Adjusting the pH within the indicated ranges is carried out using buffer compounds such as disodium phosphate. To prevent the precipitation of calcium ions introduced into water, ethylene diamine tetraacetic acid is added to the buffer as insoluble calcium salts. It is preferable to adjust the pH during the fermentation cycle by adding solutions of sodium or potassium hydrate solutions, which provide the additional advantage of reducing the viscosity of the broth without decreasing the xanthan yield and eliminating the need for buffer solution chelation. For fast fermentation, an exact amount of oxygen is necessary to ensure the growth of the bacterial culture. The fermentation medium is aerated to provide the required amount of oxygen to obtain a sulphite oxidation value in the range of 1.5 to 3.5 mmol of oxygen per liter per minute. The amount of sulfite oxidation is a measure of the acidic rate. Fermentation is carried out at a temperature of about until the concentration of xanthan in the broth reaches at least 100 ppm, preferably about 1 and most preferably about 1, k% ( 30-96 h). The viscosity of the solution is usually 4,000 cP, preferably 7,000 cP. It is desirable to kill microbial cells by adding a bactericide. A preferred bactericide is formaldehyde at a concentration of 0.210, 2 g / l, which can be added at the final stage of fermentation, before or during storage. The proposed method is suitable for working with many variants of Xanthomonas bacteria. The preferred species is Xanthomonas carnpestris. The filterability test is an experimental technique that measures the flow rate through a filter having micropores of 0.45-3.0 µm in size as a function of volume at constant pressure (2.82 kg / cm). The filtration ratio corresponds to the ratio of the time required to collect the fourth 250 ml of motility control solution to the time required to collect the first 250 ml of this solution. The filtration ratio of 1 indicates that the solution does not have a tendency to - -thation to clogging. Valid filtration ratios for mobility control solutions are 1–3 (microporous filters with a pore size of 0.45–3 µm), preferably 1.7. The required filtration ratios and pore sizes of the filters used to test a particular solution for controlling mobility depend on the permeability of the underground layers of the oil field for which the oil is being squeezed out. Solutions for mobility control based on the completed fermentation solutions obtained in accordance with the proposed method have filtration ratios that are suitable for most oil fields. If the permeability of the subterranean layers is very low, then solutions are used to control mobility with low filtration ratios, and whole or unpurified broths should preferably not have any insoluble substances with particle sizes greater than 0, b5 microns. This can be accomplished by simply storing the finished fermentation broth in the presence of formaldehyde for days. During the aging process, Xanthomonas cells are compressed in such a way that their sizes do not exceed 0 μm to 5 μm. The cells are in the form of a corkscale, and usually the largest size in length exceeds 0.65 microns and the smaller size is 0.4-1.0 microns. The storage temperature is non-critical (O135 ° C, preferably 20-45 0). In practice, storage at room temperature for a long time is carried out until a microscopic examination shows that the smallest cell size does not exceed 0.5 µm (after 3-4 days). Test methods. A gotob is 1000 ml of a solution containing 750 p. -A million xanthan in a salt solution with a concentration of 500 ppm (10: 1 NaCJ-CaCl), as follows. In a Waring type mixer equipped with a rheostat, the amount of broth (based on xanthan content) needed to obtain 0.75 g of solid xanthan is measured. Dilution is made from 1 to 6 with salt solution. Shift the mixture as follows: 40% power (2 min 60 power) 2 min, 80 power 2 min. Dilute the solution in a blender until the concentration of xanthan gum is 750 ppm and shift at 40% power per minute for 2 minutes (the solution is also used to determine the viscosity). An experimental device was used which allowed the flow rate to be determined through a microporous filter disc (47 mm, pore size 0.46-8.0 µm) as a function of volume at a constant pressure of 28 n / m. A 1 liter tank is filled with 1 l of xanthan solution (750 h.

a million), create a pressure of 28 H / M, select the Barbi valve and record the volume of the filtrate and the time (s).

The filtration ratio is determined .5

Viscosity determination. The viscosity is determined by a Brookfield synchronous viscometer (LVT model with a VL attachment). Measurements lead. at 25 ° С and speeds x 6 and 12 rpm. Viscosity is expressed in centipas,

Determination of xanthan content. Highly purified xanthanum contains about 18 glucuronic acid. The amount of glucuronic acid in the xanthan-based com-is positions is determined in the absence of formaldehyde and without borate using the Knutson and Jeans method.

The xanthan content is determined according to formula 20

Glucuronic acid,% o LLP,

., fgnp

Example 1. Xanthomonas campestris cells from oblique agar are transferred to 300 ml of UM broth, located in Z ,, 8-liter Fernbach flask, and shaken on a rotating shaker for 31 hours at 28 ° C. A 25 ml aliquot is transferred to a Fernbach flask (2.8 L) containing 500 ml of medium of the following composition, g / l:

Glucose-fructose

(Izosvit 100) 10.1

Crude Glu-3S

goat (cerelosis) 25.1

NH4N031.0

MgS04-7H iO0,10

MnS04HiO0.03

FeS04-7HxO0,0140

Waterless lemon

acid1 0

KANP04.1

KHaPL40,69

Cereose and Isosvit 100 are dissolved in distilled water and treated separately in an autoclave. The remaining ingredients are combined, adjusted to pH 6, C., and also autoclaved. The separated autoclave processing materials are then combined together.

After shaking at 28 ° C for 33 hours, 200 ml of the solution is transferred to a 4-liter orifice tank with a mechanical stirrer containing 2 liters of the medium of the following composition, g / l:

Cerelose 25.7 autoclave separately. Izosvit 100 10.1 NH4H031.0

MgS047H-iO0,10

, 03

FeS04-7H, 2.00.10

Anhydrous citric acid, 1.0 CaC1-2No O 0.20 NaHP04 3.3 0.70 Sugar is dissolved in 300 ml of water, processed separately in an autoclave, the remaining ingredients are dissolved in 1700 ml of water, processed in an autoclave, and then combined both solutions. Then, aeration is carried out at such a rate as to ensure the amount of oxygen 1 mmol / l per minute.

Fermentation is carried out at 30 ° C for 8 hours, while the pH of the solution is maintained at 5-7.5 by adding sodium phosphate-based buffer diluted with tap water. Ethylene diamine tetraacetic acid is also added to the sodium phosphate buffer to prevent calcium phosphate from precipitating. At the end of the fermentation, the viscosity of the 7ВОО cP broth (at a shear rate of 6.27), xanthan yield is 1.5.

The motility regulation solution has a filtration ratio of 1.7 (microporous filter with a pore size of 3 µm).

Example 2. The procedure of Example 1 was repeated using the following fermentation medium, g / l:

General Wednesday 1-stage Tsereloza 10,0

(NH4) T.HP042.0

KHQ, P041.0

MgS04-7HlO0,5

NZ-Amin UT (Casein Vyvarka enzyme) pH -7.0 11.0 Sowing medium (2-stage / D-glucose

Claims (6)

 (o (5 are autoclaved separately) 27.0 D-fructose 3.0 NH4H031.0 HgS04., 10 Mp504-H200.03 FeS04-7HaO0.01 Anhydrous citric acid 1.0, I, 69 Medium, to obtain the product O- glucose (processed in an autoclave separately) 27.0 O-fructose3.0 Anhydrous citric acid 1.0 NH4N031.0 Md504-7H O0.10, 03 FeS04-7H-iOp, 01 CaClQi-2H O0.20 Na HP043.3 NaN -TR040.70 Fermentation is carried out as in Example 1, except that the pH is adjusted by dissolving sodium hydroxide without simultaneously adding ethyl diaminetetraacetic acid to ethyl. Example 3. Red scale fermentation The medium is inoculated as follows: Medium for inoculum (1-stage), g / l: Cerelosis (NN4) 1 PHO MgS04-HQ O NZ-amine YT pH-7.0 Medium is introduced in portions of 500 ml, in two 2 , 8-liter Fernbach flasks. After autoclaving and cooling, the flasks are seeded with Xanthomonas campestris cult. After shaking for 3U at 28 ° C, the contents of both flasks are combined and used to seed the fermentor at 200 gallons (757, 08 p) Sowing medium (2-stage), g / l Tsereloza 10.0 (МН4) гНР042.0 КН2Р040.453, Bitter salts 0,226 NZ-amine UT pH - 7.0 4.99 Mixing fermentation medium eskoy stirrer aerated to give 1, 5 mmol of n liter of oxygen per minute. After a certain time interval, a solution of sodium oxide base is added to maintain p at 6.0-7.5. Soybean oil is added to control the formation of fine foam. After being fermented for 48 hours, the volume of the mixture, sufficient to obtain a 5 O ratio, is transferred to a 2000 gallon fermentation tank (7570 l) containing 800 gallons (3028 p) of the following medium: Sowing medium (stage NH4M03 (50% solution),, 8, kg 12.5, kg 2.15 Bitter salts, g 306 MgS04H-3.0, g G FeS04-7H O, G .30.5 Cerelose, kg 79.1 ( treated separately in an autoclave) Isosvit 100, kg 31.75 Anhydrous citric acid pH 7, O, kg 3.05 After fermentation under the above conditions for about 31 meters, the volume is sufficient to produce 10% fine volume, transferred to a 2000 gallon maternity tank (7570 L) containing 1200 gallons (4163 L). The following medium: Cerelose, kg (autoclaved separately) Isosvite 100, l (50X-HY solution), lHgdrati limestone, kg Anhydrous citric acid, kg 4.53 Na ,, kg 3.17 Bitter salts (Nd504-7NgO), g 454 MgS04-HiO, g 141 Fe504-7Ngb pH -7.0 g 45.4 Thus , fermentation ulon, obtained by known methods, provides solutions for mobility control with a concentration of 100-300 ppm of Santomonas colloid and does not contain irreducible substances ETS, chasits having dimensions exceeding 3 microns. Claims 1. Method; production of fermentation broth used to obtain motility control solutions used in oil production, involving the cultivation of Xanthomonas campestrts microorganisms under conditions of aeration on a nutrient medium containing a carbon source, preferably carbohydrates, s sources of nitrogen, phosphorus, water, and other essential trace elements, This is due to the fact that, in order to ensure the absence in the target product of practically insoluble substances, whose sizes exceed 3.0 microns, the composition of the nutrient medium Modes administered citric acid chelate calcium and iron ions in the form of iron sulfate salts in quantitative, 15 islands respectively equal 0.5-2.0; 0.02-0.07; 0,0005-0,0025 g / l volume of fermentable medium. 2.CnocoiS according to claim 1, which is also distinguished by the fact that ethylene diamide tetraacetic acid is introduced as 20 to the formation of chelate compounds. 3. Method according to claim. 1, that is, with the fact that, after cultivation, formaldehyde is introduced into the fermentation broth in an amount of 0.2-10 g / l. k. A method according to claim 1, wherein tl and h and y and the fact that as a source of nitrogen using nitrates of ammonium, sodium and potassium. 5. The method according to claim 1, wherein the water for preparing a nutrient medium contains less than 20 g / l of calcium, and other phosphates precipitating in the form of phosphates. 6. The method according to claim 1, wherein and after cultivation, the fermentation broth is kept for 3-4 days to obtain a broth that is practically free from insoluble particles exceeding the size of 0.65 microns . Priority 08/05/76 in paragraphs 2-5, 03.05.77 by paragraphs sixteen. . Sources of information taken into account in the examination 1. US patent No. 3 27226, CL, 195-31, published. 19b9.