DD149810A1 - METHOD FOR CULTURING MICROORGANISMS - Google Patents

Abstract

The invention relates to a method for the cultivation of microorganisms on CO & ind2! -H & ind2! -O & ind2 !. The invention belongs to the field of microbiological protein production and is classified in the IPK C 12 B. The object of the invention is to develop a process which enables a stable process under unsterile conditions for obtaining an egg-rich end product. At the same time a technologically optimal variant for the supply of organisms with the carbon and nitrogen source will be developed. This objective is achieved by cultivating the bacterial strain HB 201-ZIMET 612 under unsterile conditions at a temperature of 32 to 34 degrees C and a pH of 6.0 to 6.5 under normal pressure or elevated pressure. The carbon and nitrogen necessary for the growth is continuously metered into the fermenter in the form of dissolved ammonium carbonate. By this measure, the partial pressures and thus the geloest concentrations for H & ind2! and O & ind2! biomass contains up to 70% crude protein and all vital amino acids.

Description

-A-

title

Process for the cultivation of microorganisms

Field of application of the invention

The invention belongs to the field of microbiological protein production, in the IPK C 12 b.

The invention can be used in plants in which gaseous hydrocarbons are microbiologically converted by means of bacteria.

Well-known technical solutions

There are already known methods for culturing microorganisms, in particular bacteria, which grow on CO ₂ , H ₂ and air, wherein all 3 substrates are supplied in a gaseous state. / Lafferty, RM; Veding, V .; Robra, K.H. Schlegel, HG; "Permentation Technique with Oxigen Bacteria", Dechema Monographs 71 (1972), No. 1351-1369 /.

The specified fumigation ratios are in the ranges of O ₂ : CO ₂ : H ₂ = 1: 1: 8 over 2: 3: 6 to 2: 1: 7 · For biomass production are mainly microorganisms of the genera Hydrogenomonas or Alkaligenes / Schlegel, MG; Lafferty, RM: "Hovel energy and carbon sources · Production of biomass from hydrogen and carbon dioxide"; Advan. Biochem. Closely. 1 (1970), 143-68 / and Mycobacterium strains / PR-PS Hr. 2062188; Inh »Nippon Oil Co./ used ·

Pur this mesophilic organisms are optimal temperatures of 25 to 35 ⁰ C / FR -. Patent No. 2062188 "Method for obtaining new bacteria of the genus Mycobacterium" / and an optimum pH range of 6.8 to 7.2 / Lafferty, R · M .; Moser, A. j. Steiner, W ·: "The production of single-cell protein using oxyhydrogen bacteria"; Series of the GBP (June 1975) 1, pp. 87-92 /.

The gases COp, O ", Hp are absorbed by Hydrogenomonas in the ratios 1: 1.2: 4.5 to 1: 1.9: 6.6. / Ammann, ECB; Reed, L «L .; Durichek, JE Jr .: "Gas consumption and growth rate of Hydrogenomonas eutropha in continuous culture"; Appl. Microbiol. 16 (1968), 822 26 /. Specific rates of U, 0.4 b. for Hydrogenomonas / Kessler and co-workers; Mikrobiologija T. XLIV, 2, 1975, pp. 219-223; Lafferty, RM; Veding, V ·; Robra, KM; Schlegel, HG: "fermentation technology with oxyhydrogen bacteria ¹ · Dechema monographs 71 (1972) No. 1351 - 1369 /.

All of these methods have the disadvantage of employing the carbon and energy source in a gaseous state, which results in a lowering of the initial pressures for Og and Hg and, accordingly, a reduction in the dissolved amounts of these sparingly soluble gases.

Another disadvantage of the known methods is that they have to be carried out under sterile conditions. For a technical realization, this results in a high investment cost effort

Finally, in the described methods, the nitrogen supply of the organisms must be considered unfavorable. This takes place via the Hahrlösungsdosierung or the pH control with aqueous ammonia solutions. If a nitrogen compound is passed through the nutrient solution in the fermenter , resulting from the pH control with lye a high salt load of the microorganisms.

Object of the invention

The object of the invention is to develop a method which enables stable process management under unsterile conditions and optimal supply of the organisms with the C and ΪΓ sources for obtaining a protein-rich end product.

Essence of the invention

The invention is therefore based on the object of selecting and using a production culture which can be stably cultivated in the sterile process. Furthermore, the partial pressure fraction and thus the dissolved amount of the components Hp and O must be increased by suitable technological measures Increasing the gas utilization degree is achieved

According to the invention the object is achieved in that a particularly selected strain continuously on a COp-Hg-air mixture or on a mixture of Hp and air at dosage of (NBL) ₂ CO ^ and / or ЛН.HCO ^ in a strict Nutrient solution is cultured under unsterile conditions, whereby at the same time the use of (1OL) ₂ COo or · ГОдНСОо covers the supply of nitrogen to the organisms and the extent of the necessary pH control is minimized. The strain was isolated from flux mud of the Puhne (Elbe Hebenfluß) via an enrichment culture. The enrichment culture was obtained after a special selection program in a continuous process.

The strain is called HB 201 - ZIMEO? B 612 and was deposited in the depository of the IMET KuIdüren collection in the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 69 Jena, Beuthenbergstraße 11 ·

The cultivation is carried out with a nutrient medium of the following composition:

H ₃ Po ₄ (80%) H ₂ O H ₂ O 0.027 ml / 1 KH ₂ PO ₄ H ₂ O H ₂ O 0,035 g / l MgSO _4. 7 H ₂ O H ₂ O 25 mg / 1 CuSO ₄ · 5 H ₂ O 0,785 mg / 1 MhSO ₄ . 4 H ₂ O 1,825 mg / 1 PeCl ₂ . 4 H ₂ O 1,201 mg / 1 ZnCl ₂ H ₂ O 0,322 mg / 1 CoSO ₄ . 7 , 18 0,036 mg / 1 UiSO ₄ . 7 , 4 0.109 mg / 1 Al ₂ (SO ₄ > 3 2 0,186 mg / 1 Ca (NO ₃ ) 2 0.883 mgA Na ₂ MoO ₄ , 6 0,041 mg / 1 H ₃ BO ₃ 1,286 mgA CrCl ₃ 0.077 mg / 1

These salts are sufficient for 1 g biomass dry substance, to obtain higher biomass concentrations, the nutrient solution is balanced in proportion to the biomass increase higher · The supply of organisms with the C and N source via the dosage of a solution of (NH ₄ ) ₂ C0o or NH ₄ HCO ₃ , whose concentration is adjusted according to the productivity of the process so that the growth of organisms is not limited.

Due to the amphoteric behavior of an aqueous ammonium carbonate solution, the pH of the culture medium remains within the interval desired for the fermentation and pH control is largely unnecessary.

The main type selected by conventional selection methods from the enrichment culture, strain HB 201, is characterized by:

Cell Morphology:

colony morphology

Physiological behavior:

The strain HB 201 is a weak to strong vibrioid curved movable rods (sometimes forming up to 11/2 spiral turns) whose size is 0.5 to 0.8 χ 1 to 2.5 / um. The cells often appear as diploforms and react gram-negatively · At the cell poles are often phase contrast optically denser than the rest of the plasma appearing deposits that do not always respond lipophilic · The lipophilic deposits are often in the plasma ·

Tray 3 to 5 days of incubation at 30 to 32 ⁰ C form cream-colored to light beige, circular, smooth-edged, convex colonies with a moist shiny surface and buttery greasy consistency of 0.5 to 1 mm diameter ·

Since growth on Peρton - Glukoseagar is very low, the data on mineral agar are C0 ₂ / H ₂ / air (or · Op) atmospheres.

The strain grows autotrophic on mineral media when the gas atmosphere contains H ₂ »CO ₂ and O ₂ or air. Perner, he can grow on the following substances as sole carbon source: acetate, succinate, pyruvate, malonate, acetamide and ethanol. In contrast, the following C sources are used (as sole C source)

Taxonomic classification:

Other features:

no growth: glucose, fructose, galactose, mannose, cellobiose, maltose, rhamnose, D-ribose, D-xylose, mannitol, m-inositol, L-alanine, L-valine, L-tryptophan, L-serine and asparagine · Der Stamm reacted catalase-positive and oxidase-negative «

Due to some feature combinations is not an exact classification to mountains? (1974) possible. Most likely, the assignment to Part 6 (Spiral and Curved Bacteria) appears.

pH 5.5 to 7.2, optimally 6.0 to 6.5 range, temperature range 28 to 36 ⁰ C, optimal 32-34 ⁰ C, Begasungsverhältnis CO _2: O _2: H ₂ = 1: 1, 86: 5.56 to 1: 2.6

'2 * ^W 2 7,78.

The maximum growth rate of the microorganism HB 201 is / I _013x = 0.18 h. Under non-sterile conditions, this organism is in the temperature range of 28 to 36 ⁰ C, preferably at 32 to 34 ⁰ C, at a pH in the range of 5.5 to 7.2, preferably 6.0 to 6.5, and A pressure of 1 to 15 atm can be cultivated continuously at residence times of a minimum of 5.5 h. · Slime-free fermentation is possible in an unsterile process · By lowering the pH to 5.5, an almost complete culture purity is achieved (95 % of the culture) Main type by cell number, corresponds to> 95 % biomass fraction).

The fumigation takes place with carbon dioxide-hydrogen-air

H,

1: 1.86: 5.56 to

Mixtures of COp: Op

1: 2.6: 7.78.

The nutrient solution is prepared with tap water and adjusted with phosphoric acid to a pH of ^ 3.

The concentrations of the components of the nutrient solution are chosen so that the Uährsalzangebot always the

current biomass concentration corresponds or only slightly deviates from it. The end product of the process is a dry matter containing up to 70% crude protein and all vital amino acids. The following examples illustrate the invention.

Example 1:

In a stirred fermentor, 1 kg of culture broth HB 201 - ZBiET B 612 with a concentration of 2 to 3 g / l is used. The temperature is set to 34 ^° C. and the residence time to 10 h. The medium is gassed with Hg, CO ₂ and air in a ratio of CO ₂ : O ₂ : H ₂ 1: 1.86: 5.56.

The regulation of the pH is carried out with a 1% ammonia solution; This ensures at the same time the nitrogen supply of the organisms. The nutrient salts necessary for growth are metered into the per- mentor as a uniform nutrient solution which is brought to a pH value by addition of HCl. After the biomass has grown to a concentration of about 4 g / l, the residence time is increased Lowered 6 hours. The nutrient solution is always adapted to the current biomass concentration and overbalanced for 0.5 g BTS / 1 · The nitrogen concentration of the culture medium is maintained between 50 and 150 mg / l · Under these conditions, biomass concentrations of 3.3 g / l become stable in the continuous process achieved a productivity of 0.55 g / l · h; the crude protein content of the biomass is 64.4 %.

Example 2;

1 kg of seed material of the culture HB 201 - ZIMET B 612 with a concentration of 2 to 3 g / l is used in a stirred fermenter.

The temperature is adjusted to 34 ⁰ C and the residence time to 10 h. The medium is gassed with H ₂ , CO "and air in a ratio of CO ₂ : O ₂ : H ₂ = 1: 1.86 ι 5.56. The regulation of the pH is carried out with a 1% ammonia solution; This ensures at the same time the supply of nitrogen to the organisms. The nutrient salts necessary for the growth are dosed into the per- mentor as a uniform nutrient solution, which is brought to a pH value of 3 by the addition of HCl. "After the biomass has been concentrated to a concentration of approx 4 gA is increased, the residence time is reduced to 6 h · The nutrient solution is always adapted to the current biomass and over-balanced for 0.5 g BTS / 1. Subsequently, the CO ₂ gassing is turned off and dosed a (UHj) ₂ C0o solution in the fermentor. The total fumigation intensity is not changed, the O ₂ and H ₂ partial pressures are increased at the expense of CO ₂ in the stoichiometric ratio.

The metered (UH,) ₂ C0 ₃ amount corresponds to 900 mg / 1 · h (calculated as CO ₂ ),

After setting a pH of 6.8, there is no need for additional pH regulation and ΪΓ supply.

Under these conditions, biomass concentrations of 3.7 g / l corresponding to a productivity of 0.61 g / lh are achieved in the stable continuous process; the crude protein content of the biomass is 62.5 % ·

Claims (1)

invention claim Process for the cultivation of bacteria on CO ₂ -H ₂ in the presence of an aqueous mineral salt solution and a gas containing free oxygen, characterized in that the bacterial strain HB 201 - ZIMET B 612 is cultivated continuously under preferably unsterile conditions, whereby the C- and Η- Source in porin an aqueous solution of (NH.,) _O C0 _o and / or KH, HCO- is metered into the per- mentor.