DD283738A7 - METHOD FOR CULTURING MICROORGANISMS - Google Patents

Abstract

The invention relates to a process for the cultivation of microorganisms on methane / natural gas as the sole carbon source. According to the invention, the novel methanotrophic bacterial strain IMET 11334 and the methanotrophic bacterial strain ZIMET B 502 are cultured together as a defined mixed culture continuously on a methane-air mixture under normal or elevated pressure in a strictly balanced nutrient solution under unsterile conditions. The final product obtained by these methods contains up to 75% crude protein and all vital amino acids. {Cultivation; Eiweiszgewinnung; Bacteria; crude protein; microorganisms; Prozeszfuehrung; methanotroph; Amino acids; Methane; Mixed culture; unsterile}

Description

Characteristic of the known technical solutions

There are methods for the cultivation of microorganisms, in particular bacteria, based on gaseous hydrocarbons, eg. As methane, known. Thus, US Pat. No. 4,042,458 describes a process in which a strain of methylococcus is raised to methane in a mixed culture with heterotrophic and methylotrophic microorganisms which assimilate metabolic products with a productivity of 0.9 g / lh. The recovered biomass contains 69% crude protein. DE-OS 2307692 and DE-OS 2241258 also describe methods with a Stemm Methylococcus. The disadvantage of these methods lies in the low productivity, low crude protein content and sensitivity to high ammonium ion concentrations. Also known is the growth of a symbiotic bacterial culture (SU-US 460745), at a flow rate to 0.16 h " ¹ and a temperature of 33 ⁰ C and a pH of 6.7 on natural gas. A thermotolerant strain of the genus Methylococcus capsulatus , which grows at a temperature optimum of 38-39 ⁰ C and a flow rate of D = 0.12 h " ¹ , is described in SU-US 467620. The productivity achieved with this culture is 1.2 g / kg · h. The above-mentioned methods and the methods described in the patents (GB-PS 1421135, GB-PS 1397735, DD-PS113928, DD-PS 124194) share the following disadvantages:

- relatively low growth rates

- low productivity

- low tolerance levels with respect to ammonium nitrogen concentration.

In DD-PS 157866 a method is presented, which is characterized by high productivity at high flow rates. The disadvantage of the dipres method is based on the known for bacteria of this genus high sensitivity to Ammoniumstict'StoiTKcnzentrationen. The microorganisms described in patent DD-PS148465 tolerate ammonium nitrogen concentrations up to 400 mg / l without loss of productivity, but the flow rates achieved do not meet the requirements of a production culture to achieve high productivity.

Get the invention

The invention aims to develop a process that ensures stable process control under non-sterile conditions with high flow rates while ensuring a protein-rich end product.

Explanation of the essence of the invention

The invention has for its object to select and use a production culture that grows in the unsterile process with high biomass concentrations and compared to the previously known methylotrophic microorganisms has a high growth rate and a sufficient selection advantage and high competitiveness.

According to the invention the object is achieved in that the new methanotrophic bacterial strain IMET11334 is cultured together with the methanotrophic bacterial strain ZIMET B 502 as a defined mixed culture continuously on a methane-air mixture under normal or elevated pressure in a strictly balanced nutrient solution under unsterile conditions.

The new strain IMET11334 was deposited in the strain collection of the central institute for microbiology and experimental therapy of the AdW of the GDR. It is characterized by the following characteristics:

Cell Morphology:

short thick to medium sized straight rods with a size of 0.8 to 0.9 x 1.2 to 2.0mm. The cells predominantly appear as a diploform, are gram-negative and immobile. During cultivation under normal conditions in a shake flask, no sudanophilic granules were stored in the cells. Slime formation was not observed.

Colony morphology:

On mineral agar plates, after 2 weeks, reddish brown ir of the middle, dark brown, flat, fain amorphous colonies are formed with a glossy margin when an inorganic nitrogen jellyfish and methane are offered as C source. The diameter of the colonies is about 1 mm, the color intensity of the colonies depends nt> r.h the growth period. After 8 days of incubation, the colonies are yellow-orange to light brown and reddish brown after 2 weeks.

Waltere features: No growth on peptone-rich media Temperature range: 20 to 42 ⁰ C

pH range: 4.7 to 6.9

Tolerance limit for ammonium nitrogen: 600 mg / 1 Optimal growth occurs at 100-200 mg / l. The strain ZIMET B 502 forms the base strain in mixed culture and has already been described in detail in DD-PS148465

described. The strain IMET11334 is serologically distinguishable from the strain ZIMET B 502. The strain reacts negatively with Kaninchan antiserum of strain ZIMET B 502 in both slide agglutination and challenge

Immunofloureszenz. The ferment jrbiozö nose consisting of 70-95% of strain ZIMET B 602 and 5-30% of ^c . 'Ammes IMET11334, has the advantage over the previous cultivation of only one methanotrophic strain (ZIMET B 502) that

in the process of methane conversion, two defined methanotrophic bacterial strains with different

Metabolic pathways are involved, with a maximum growth rate of M _max = 0.33 h ~ 'is achieved. The symbiotic Relationships of both methanotrophic strains allow this high rate of growth. Furthermore, that is Presence of two methanotrophic strains with different metabolic pathway in the event that benefits

possible infestation of virulent phages dia mass cell cultivation disturbs because only one strain would be affected.

Fermenting the strains ZIMET B 502 and IMET11334 as a defined mixed culture increases productivity

by 25% compared to the fermentation with the strain ZIMET B 502. Under non-sterile process conditions sinddie organisms ZIMET B 502 and IMET11334 in the temperature range of 28 to 42 ⁰ C, preferably at 38 to 4O ⁰ C, at a pH in the range of 4.7 to 6.8, preferably 5.4 to 6, 2 and a pressure of 0.1 to 1.0 MPa continuously culturable at residence times to a minimum of 3.5h.

Fumigation takes place with methane-air mixtures of 1: 3 to 1: 6. The regulation of the pH is preferred with a Ammonia solution made. The nutrient solution is prepared with tap water and with phosphoric acid to a pH value

set by s 3. In this case, concentrations of the constituents of the nutrient solution are selected so that the nutrient salt supply always corresponds to the actual biomass concentration or deviates only insignificantly from it. The end product of the process is one

Dry matter containing up to 75% crude protein and having all vital amino acids. . The invention is explained in more detail in the following examples: example 1

In a stirred fermentor, 5 kg of precultivated seed of the culture mixture ZIMET B 602 (85% of the methane assimilators) / IMET 11334 (15% of the methane assimilers) with a concentration of 8 to 10 g / l are used. The nutrient medium contains distilled water per liter:

H ₂ PO ₄ (80%) 0.028 ml KH ₂ PO ₄ 35 mg MgSO ₄ .7H ₂ O 25 mg CuSU ₄ * 5H ₂ O 0.785 mg MnSO ₄ * 4H ₂ O 1.825 mg FeCl ₂ * 4H ₂ O 1.2 mg ZnCl ₂ 0.322 mg CoSO ₄ .7H ₂ O 0.036 mg NiSO ₄ .7H ₂ O 0.109 mg Al ₂ (SO ₄ J ₃ * 18H ₂ O 0.186 mg Ca (NOj) ₂ * 4H ₂ O 0.883 mg Na ₂ McO ₄ * 2H ₂ O 0.041 mg H ₃ BO ₃ 1.286 mg CrCl ₃ * 6H ₂ O 0.077 mg

The temperature is set at 38 ^° C., the pH at 5.7, and the residence time at 6 hours. The medium is gassed with 15 l / kg · h of methane and 60 l / kg · h of air. The control of the pH is carried out with a 2% ammonia solution; At the same time the supply of nitrogen to the microorganisms is ensured. The nutrient solution is first balanced to a bacterial cost of 15g / l. After the biomass has grown to a concentration of 15 g / l, the residence time is reduced to 4 hours. The ammonium nitrogen concentration is kept between 60 and 300 mg / l. Under these conditions, in the stable continuous process, bacterial concentrations of 12 g / l, corresponding to a productivity of 3.0 g / kg · hr, are achieved. The proportion of methanotrophic strains in the mixed culture is 85% ZIMET B 502 and 15% IMET11334. The crude protein content of the biomass is 75%.

Example 2

The culture of the bacterial mixed culture is carried out as described in Example 1, but the temperature is set to 32 ⁰ C, the indwelling tent to 6 h. Under these conditions a biomass concentration of 14g / l is obtained in the stable continuous process. This corresponds to a productivity of 2.3g / kg · h. The ratio of the methanotrophic strains of the mixed culture is based on 70% ZIMET B 502: 30% IMET11334.

Example 3

The cultivation of the seed culture is carried out as described in Example 1, but the pH is adjusted to 5.0 and the residence time to 8 h. In the stable continuous process, a biomass concentration of 16 g / l arises under these conditions. Thus, a productivity of 2.0g / l · h is achieved. The proportion of methanotrophic strains in these culturing conditions is 95% ZIMET B 502 and 5% IMET11334.

Claims (1)

Process for the cultivation of microorganisms on methane / natural gas as sole carbon source in the presence of an aqueous nutrient salt solution, a gas containing free oxygen and the methanotrophic bacterium strain ZIMET B 502, at a temperature of 28 to 42 ⁰ C, a pH of 4.7 to 6.8 and a pressure of 0.1 to 1.0 MPa, characterized in that the new methanotrophic bacterial strain IMET11334 is cultured continuously together with the methanotrophic bacterial strain ZIMET B 502 as a mixed culture, wherein the proportion of the strain IMET11334 within the mixed culture 5 to 30 % is. Field of application of the invention The invention belongs to the field of microbiological protein production and can be used in plants in which gaseous hydrocarbons are microbiologically reacted with the aid of bacteria.