SU667154A3 - Method of obtaining biomass - Google Patents

Description

one

The invention relates to a technique for producing biomass on a nutrient medium containing methane as a carbon source.

A known method for producing biomass involves growing a mixed culture of bacteria on a nutrient medium containing methane as a carbon source, a nitrogen source and necessary mineral salts, under conditions of aeration with a gaseous mixture containing free oxygen and methane using methane-consuming microorganisms. bacteria from the genera Methy lomonas, Methylobacter, Methylosinus or Methylococcus 1.

The disadvantage of this method is the increased formation of foam in the growth process.

The aim of the invention is to reduce foaming during the growth of microorganisms.

This is achieved by the fact that in the proposed method for producing biomass, a culture of bacteria consuming methane is simultaneously grown with one or a mixture of bacterial cultures from the genera Acinetobacter,, AIcaIigenes, Agrobacteriuirt Mcuraxella

Pseudomonas, Flavobacterium, Bacillus, Miterocpccus ijJiH Pedigcpcciis do not consume methane and use culture of bacteria that consume metabolic products of metabolic products that consume methane.

In this case, bacteria of the species Methylococcus capsulatus are used as a culture consuming metane.

In addition, the NCIB strain (110,856 M. capsulatus) is used as a culture that uses methane.

In addition, in 1 quality of the culture consuming methane, the ATCC strain No. 19069 was used. M. Capulatus. At the same time, as a culture,

Foaming agents are used / Moraxella NCIB No. 11135 strain is used.

In addition, Alcaligenes, Agro- .bacterium or Pseudpmonas NCIB® shzb strain is used as a culture consuming foaming agents.

In addition, Alcaligenes, bacterium or Pseudomonas NClg W11137 are used as a culture consuming foaming agents.

In addition, as a culture that uses foaming agents. The strain Alcaligenes or P seudpmonas NCIB No. 11138 is used. In addition, AlcaiigenesNCIB No. 11139 strain was used as a culture consuming foaming agents. In addition, Acinetobacter NCIB 7win4o is used as a culture that consumes pricing substances. - ---. - ..,. .- - .., .... v, .--- ™ -... - And also, besides ttego, the strain Mcaligenes, Agrobacterium or, P seudpmonas NC is used as a culture that consumes foam. IB No. 11141. In this process, the growth is carried out at pH 5.5-7.0 and 305,. -. In addition, the concentration of am ions in the nutrient medium during the growth process is maintained at 2-50 mg / l. The method is carried out as follows. Mixed cultures of bacteria from Methyioinuhas, MethyIobacter, Methyiosinus or Methylococcus genes are grown on a nutrient medium containing methane as a carbon source, a nitrogen exchanger and a necessary mineral mineral. - -. In order to reduce the foaming during the process of growing, a culture of bacteria that treats cubic acid cubes with one or a mixture of bacterium cultures from Acinetobacter, Alcaligenes, Agrobacterium, Mbraxella, Pceudomohas, Flavobacterium, Bacillus, Micrococcus or Pedrococarium, Pceudomohas, Flavobacterium, Bacillus, Micrococcus or Cyclicromatum, is treated. cultures of bacteria that cause methane. At the same time, the cultures consuming methane are used by the bacterium of the species MethyTocddcus capsubatus, strain NCIB No. 10856 M. capsulatus, strain ATCC No. 19069 M. capsulatus -; As crops consuming H-fossi-hc-thWeWeWTigGbTGblSH-shtalsh Moraxella NCIS No. 11135, NCIB. : No. 11136, NCIB No. 11137, NCIB No. 11138 and Acinetobacter NCIB No. 11140, Alcaiigenes NCIB No. 11139 and NCIB No. 11141 strain. The leveling process is carried out at pH 5.5-7.0 and 30-55s, and the concentration is ammonium in the nutrient medium in the process of growing; support 2-50 mg / l. The process is carried out under conditions of aeration with a gaseous mixture containing bore oxygen and methane, while the amount of methane is usually 16 volumes to 84 volumes of air, it can also be from 8 to 40, the volume of air is 92-60 volumes of air. meper 1. A mixture of bacteria strain ATSS 19069 Methylococcus capsu. latus, consuming methane and elemental nitrogen, and bacteria, which consume foam-forming substances formed by these bacteria, are cultivated in 5 liters (working volume) of broth contained in a seven-liter fermenter. The process is not aseptic, the temperature is maintained, and the stirring is carried out with a mixer at a rotation speed of 1000 rpm. Add broth to the broth with a dilution degree of 0.18 hours of the following composition, mg; KN.NRO 175, 0; Nag.HPO. 12 - 150.0; MgS04-7H20 - 85.0; CaCI. (Anhydrous) 20, 0; CUS04-5H20 - 20.0; Geozod7N, O 7, 5; 2PZOD, 7H O - 0.5; 0, 5; CoCl26H2 0 - 0.02; Na "MoO ,. 2H2O0, 2; ,; distilled water up to 1 l. The pH is adjusted to 6.4 using 2.0N sodium hydroxide solution, nitrogen is introduced in the form of ammonium hydroxide and air, while the amount of ammonium hydroxide is injected to meet the nitrogen requirement of the bacteria present by 95% and the remaining 5% satisfy nitrogen air. Methane is fed at a speed of 6 v / v "hour and vozuh -. at a speed of 24 rev / rev. Fermentation is stable, while receiving a cell density of 3.4 g / l with a performance of 0.61 g / l hour. . In all examples, cell weight is expressed in terms of dry weight. The formation of foam does not detect bacteria. Then the concentration of the aqueous nutrient medium and ammonium hydroxide is double. This leads to an increase in cell density up to 6.5 g / li of productivity up to 1.7 g / l per hour. No foam is formed. Bacteria present in the broth consist of 3 85% of the cells of the ATCC strain 19065, bacteria of Methylococcus capsulatus, which consume methane and elemental nitrogen, and 15% of the mixtures of bacteria that do not consume methane. The latter was isolated by diluting the broth sample to 10, applying the diluted sample on the agar in a petri dish and incubating for 48 hours under aerobic conditions in the absence of methane. Separate colonies were removed: agar. And continuously seeded in fresh medium to obtain a pure culture. The cultures obtained were listed in the national collection of industrial bacteria Aberden; (Scotland) under the numbers: 11135, 11136, 11137, 11138, 11139, - 11140 and 11141. Then the bacteria are identified in accordance with the existing classification. The data are given in table.1. The mixture of methane-consuming bacteria consists of 3% of unidentified cells of g4) of amplexically-changing bacteria in the form of sticks Strain NCIB No. 11134 and 12% of a mixture consisting of

MoraxellaNCIB 11135 strain, strain Alcaligenes, Pseudomonas or Agror bacterium NCIB.iri36, Pseudomonas or strain Alcaligenes NCIB W11138 strain Acinetobacter NCIB №11139, rtamma Acinetobacter NCIB №11140, a strain of Pseudomonas, Alcaligenes or Ayrobac-terium NCIB №11141.

For comparison, the process is carried out under cultivation under aseptic conditions in the presence of microorganisms consuming methane and elemental nitrogen, Methylococcus capsulatus ATCC 1 19069. The process is carried out continuously at a dilution rate of 0, at a cell density of 3.3 g / l, and productivity of 0,59 g / l hour.

Fermentation is unstable due to foam formation by culture, which leads to difficulties in controlling the level in the fermenter and achieving stability of the process. The foam also reduces the volumetric penetration ability so that the resulting substance is not continuously withdrawn.

Then the concentration of the nutrient medium is doubled, which leads to an increase in cell density of up to 5.5 g / l and abundant foam formation, followed by termination of the process. Characteristics of the strains are given in table.1.

Characteristics of other strains are given in table 2.

Table 1

Microscopes Morphologists GDSA Staining Gram Me Me O / F Glucose Motility Argenin Ammonium C. Ahmalna Ammonium Sugar-Ethano

Tween 80 Hydrolysis Sticks 0.35-0.5 x 1l, 25ju rounded ends, edges parallel, some slightly curved, isolated, some chains contain up to 8 cells. Round, smooth, lustrous, diameter of a rounded protrusion 2-3 mm, edge c, broad, beige with yellow mid-weight. Like GDSA, NA and Acupuncture with gray inclusions. O sticks, 35-0.5 x, 25-1.5 / J parallel sides, single. Round, glossy, shiny, small curvature when pricked with a needle, the edge is solid, translucent light brown, large colonies on agar. Like GDSA, but with a diameter of 1 mm.

Tapping Gelatin ONG1G Oxidae Cup Filter Boomers Growth Naagare Mac Conca Malonag

Growth in cyanide potassium

Ureaza Kaeinaza Catalaza

T: spur hydrogen Indole Acetylmethyl arbi nol

The recovery of

Sensitivity to nicillin 2.5 immun units

Anaerobic growth of SDS

Mf agar + 0.2% glitch

Growth on araps C in 50% methane

Growth on MC Agar in Butanol Vapors

| "11 in acetone vapors

-I ". "| | in methanol vapor

| 1 1 in pairs; fl; anrla. . . || "T in pairs

 - - -

„I - in Larah

formaldehyde

Growth on MS Agar in Gramuic acid

". agar 0.2% (in glucose

667154

eight

Continuation of table 1

+ f

+ +

--in liquid MS without additives

- plus 0.2% glucose

 plus 0.2% arabinose

- plus0,2% galactose

- plus0,2% lactose

- plus0,2% xylose

- plus0,2% sucrose

- plus 0.2% mannitol

- plus0,2% glycerol

- plus0,2% salicin

- plus0,2 + laculoses

Presence of flagella

(electron microscopic)

Sporulism in manganese medium Mikrbsko-Sticks Sticks 0.35-0.5 x 0.5 l IfS 0.5 x 1.5 p x 1.25, rounded single species. round ends, pa- or parallel parallel, multiple ends, single. or mated

Round, smooth, glossy, convexity of 1-2 mm is small, diameter is 2-3 mm, edge

cool, beige

H N N

1 side

iT a b l and c aa 2 Short short sticks, Pdeomorphic Pleomorph sticks, 0.35 x chopsticks, 1 rounded x 1.75 rounded ends,. the sides of the edge are not parallel parallel parallel, uniform

eleven

Neregul p-Variable spot staining, bipolar

dark

chink

crack

(+)

.

+

667154

12

Continuation of table 2

VariableVariable

irregular p-dyed coloring

Variable Variable Variable irregular p-irregular p-irregular coloration staining stitching

chink

chink

chink

(+)

+ +

+ +

Hydrogen sulphide

Indole

Acetylmethylcarbins

Nitrate reduction

Penicillin sensitivity of 2.5 immune units

Anaerobic growth GDSA

Ms agar + 0.2% glucose

Growth on MS Agar in 50% Methane.

Growth on MS agar in butanol vapors - - in acetone vapors

- - in methanol vapor

- in ethanol vapor

- in ethane vapor

- in formaldehyde vapors

Growth on MS in formic acid vapors

15

 : rm-:;

a plus

0.2% arabinose

- plus, 0.2% ha. lactose

-. "".

a plus

about 2% lactose

0.2% xilbzy I

plus 0.2% sucrose.

- Wh.

a plus

0.2% mannitol

A PLUS

oh 2 "glycerol

plus of2% salicin

-. ""

plume

0.2% levlosa

667154

sixteen

Continuation of table 2

f

o-f-oxidation / fermentation

GDSA glucoeo-yeast bulk agar: glucose - 5 g, peptone - 10 g, yeast extract - 5 g,; rennet - 10 g, agar - 20 g, distilled water - 1000 ml

Autoclaving with iS f№H.

PI - nutrient agar (oxo SMZ)

MS is a mineral medium (liquid or solid) when JO is on a sloping nutrient agar. The base of the liquid slanted agar is placed in sterile distilled water. Culture growth after incubation is assessed at the air-water interface by means of faeo-concentration microscopy. Manganese medium for sporulation of a plate with oblique nutrient agar to which 2 mg / ml of Mn ions in the form of manganese sulfate is added. Cultures are incubated for up to 14 days at 30 ° C and then inoculated with spores. Cultures were also evaluated for sperm capacity after exposure to a temperature of 80 ° C for 10 minutes. Example 2. In a cTepH bHSft epo di 7 liter tank, 4500 ml of sterile aqueous feed solution is placed. The composition of the aqueous feed medium, as described in Method 1, with the addition of: 1.770 g / l of nitric acid. 500 ml of actively growing culture of microorganisms Methylococcus capsulatus ATSS No. 19069 using methane and elemental nitrogen are introduced into the fermentation tank and ensure its periodic operation under aseptic conditions at a stirring speed of 500 rpm, aeration rate of 10 revolutions per hour, and methane consumption 10 revolutions per hour, the pH is maintained at 6.5 by the addition of 1.0 g / l sodium chloride hydroxide solution or 3.6 n. solution of sulfuric acid in terms of conditions. When the cells reach a density of 1.0 g / l, the agitation speed is increased to 1000 rpm. Then the operation of the vat is switched to continuous mode at a dilution rate of 0.05. After 12 hours of continuous operation, the dilution rate is increased to. About. / about an hour, and consumption of methane -: About.:. rb / bb hour. Eirie after 15 hours; skEE) Ost% dilution increased to 0.18 hours; B1xZtnost cells constituted e; 3 iv, and the rate of formations of Yishlerno 0.59 g / l -h. r.ozhim.,% of work is unstable due to foam,, p; 4pL.zuyuschey with the growth of culture. Foam prevents stationary operation. At this stage, the aseptic conditions are violated, and after two hours of non-septic tank work, the jaaHHe foam decreases and some types of bacteria surround the culture. In 24 hours the mode of operation is established when the cell density is 3.4 g / l, and the formation rate is 0.61 g / l h. Foaming is no longer observed. By analyzing the broth culture, a bacterial culture is detected as in Example 1. Example 2 shows that foam formation can be effectively reduced in the presence of a mixture of microorganisms. Approx. 3. 4.5 liters of aqueous nutrient medium of the following composition, mg, are placed in a seven-liter fermentation bath, mg: HNGj - 3500.0; KN, PO.- 350.0; NajHPQ,} 12H20 - 300.0; MgSO. 7H20 170 CaCEj (anhydrous) - 40.0; -4.0; Feso. CuSO -L j 2 1 f -f, i: t: j 7 H 15.0; ZnS047H20 - 1.0; SoSebbN.O - 0.04; Mn304-Hz, O - 1.0; Na2MoO -2 H2O - 0.4; distilled water up to 1 l. The pH of the medium was adjusted to 6.4 by the addition of a 2.0N aqueous sodium hydroxide solution, then 500 ml of the ahyptically grown Methylococcus capsuIatus ATSS No. 19069 cultured under aseptic conditions was introduced into the fermentation tank and ensured its operation in a periodic mode with a stirring speed of 1000 vol / min at 45С, air consumption - 10 rev / h and methane - 10 rev / rev. The pH of the medium is maintained at 6.4 by the addition of a 2.0 N aqueous solution of sodium hydroxide or 3.6 g of a solution of sulfuric acid using an automatic titration device. Upon reaching a cell density of 2.0 g / l, the mode of operation is transferred to a continuous, injecting an aqueous nutrient medium, HenpejjfcJBHo, into a broth culture and discharging it at a rate that ensures the stability of the working volume in the fermentation tank with a volume of 5 liters. The initial dilution rate is 0.05 hour. As the cell density increases, the mixing rate, the consumption of methane and the air consumption gradually increase after 36 hours, the dilution rate is 0.18 hours, the mixing speed is 2.000 rpm, the air consumption is 48 vol / vol. H and the consumption of methane is 12 v / v “hour. At this stage, an aqueous nutrient medium is added to an aqueous solution not containing nitric acid. The new solution has the following composition, mg: - 700; NDRO12H, 0 600; - 340; CaCIj Gbezvodny) - 80; Czzbd. - eight; , jO- 30; - 2; MPzod NZO 2; CoCl2 6Hj, 0-0.8; Ka2MoOD2NdO 0, 8; distilled water up to 1 l. Nitrogen from the form of ammonium hydroxide in the amount of 95% is added to the broth culture separately for the growth of microorganisms. The rate of administration is determined by establishing the density of the cells of the growing culture (optical density method) and using the density of the cells to measure the rate of nitrogen binding to the biomass. The remaining 5% of nitrogen is introduced together with the elemental nitrogen of the air supplied to the fermentation tank. After another 60 hours, a steady state is reached at a cell density of 11.67 g (dry weight) per liter and a formation rate of 2.1 g / l hour. With the growth of a culture of foaming, it is not observed. Bacterial analysis of the broth culture shows the presence of the same species as in Example 1. Example 4. Methylococcus capsulatus NCIB No.10856 was completely repeated as described in the example. New isolate from soil sample. The steady state of the process is achieved at a cell density of 13.14 g (dry) weight per liter / hour, which corresponds to a formation rate of 2.4 g / liter per hour. Foam formation is not observed; the bacteria species is similar to that of Example 1, except that the methane-using microorganism type is Methylococcus capsulatus NCIB No.10856.

The concentration of biomass in the broth culture entering the centrifuge, g. Weight of dry cells per liter

The concentration of biomass in the stream coming out of the centrifuge, weight of dry cells per liter

The amount of biomass from 1 l of broth culture, weight of dry cells per

From the results of Table 3 it can be seen that biomass is efficiently extracted by centrifuging a broth culture containing the new microorganism Methylococcus capsulatus NCIB No 10856, while only 97.5% of the biomass is removed from the broth culture containing the known AATS strain 19090.

From the presented data it is also seen that under equal conditions, the productivity in the case of using the new NCIB strain No. 10856 is much higher compared to the known ATCC strain No. 19069.

In addition, a new type of steel has a higher methane yield compared to the known one.

10.50

12.90

13,14

0,0015

0,0015 0,26

The new species Methylococcus capsulatus NCIB No.10856 forms less than 50. The amount of encapsulated polysaccharide materials per unit weight of biomass compared with known species. - - - 55

Claims (14)

1. A method of producing biomass, involving the cultivation of a mixed culture of bacteria on nutrient 60 medium containing methane as a carbon source, a source of nitrogen and non-salable mineral salts; x aerating with a gaseous mixture containing free oxygen and methane, using as 65 A stationary-grown broth from the fermentation tank is placed in the center 1 of a fugu of continuous action (Sharpless type 16) with a load of 17 kg, centrifugation is carried out at two different flow rates of 5571 ml / min and 774 ml / min. The concentration of bomoma ssy TG pbtkёtT outgoing from the centrifuge is measured in terms of the weight of dry cells per liter. Then, based on the weight of the dry cells, the weight of the biomass leaving the centrifuge is determined. Comparative data of centrifugation of cultures containing Methylococcus capsulatus ATSS No. 19069 (example 3) and Methylococcus Capsulatus NCIB W10856 (example 4) strains are shown in Table 3. That l and c Methane-consuming microorganisms of bacteria from the genera Methylomonas, l ethyiobacter, Methylosinus or MethylQcoccus are distinguished by the fact that, in order to reduce foaming in the process of growth, bacteria genera Acinetobacter, Alcaligenes, Agrobacterium, Moraxella, Pseudomonas, Flavobacterium. Bacillus, Mibrocqccus or Pediococcus that do not consume methane and sputlating foaming metabolic products of a culture of bacteria consuming methane, 2. Method according to n.i, which is related to the fact that the quality of the culture consuming methane is used by bacteria of the species Methylococcus capsulatus. 3. Method according to paragraphs. 1.2, I differ from the fact that NCIB 1 10856 M.capsulatus is used as a culture consuming methane. 4. Method according to paragraphs. 1.2, characterized in that the ATCC 19069 M.capsulatus strain is used as a culture consuming methane. 5. Method according to paragraphs. 1-4, which is such that Moraxella NCIB No. 11135 is used as a culture consuming peioobrazuyuschie substances. 6. Method according to paragraphs. 1-4, about t l and They also use the strain Alcaligenes, Agrobacteriuin or Pseudomonas NCIB 11136 as a culture that consumes foaming substances. , 7. The method according to paragraphs. 1-4, about tl ich and you n and also with the fact that in quality cultures consuming foaming agents use a strain of Alcaligenes, Agrobacterium or Pseudomonas NCIB No. 11137. 8. Method according to paragraphs. 1-4, characterized in that Alcaligenes or Pseudomonas NCIB No. 11138 is used as a culture consuming foaming agents. 9. Method according to paragraphs. 1-4, about tl and ch and y and with the fact that, as the culture consuming foaming substances, the strain Alcaligenes NCIB no. 11139 is used. IP. The method according to paragraphs. 1-4, of which they are used, and that Acinetobacter NCIB No. 11140 is used as a culture consuming foaming substances. 11. Method according to claims; 1-4, differing from the fact that the culture that consumes blowing agents is Alcaligenes, Agrobacterium or Pseudomonas NCIB No. 11141. 12. Method according to paragraphs. 1-11, on the basis of the fact that the growing process is carried out at a pH of 5.5-7.0. 13. Method according to paragraphs. 1-12, characterized in that the growing process is carried out at 30-55 14. Method according to paragraphs. 1-13, about tl and tacho that the concentration of ammonium ions in the nutrient medium in the process of growing support 2-50 mg / l. Sources of information taken into account in the examination 1. USSR author's certificate No. 421199, cl. C 12 D 13/06, 1974.