RU2822163C1 - Method of producing carotenoid-enriched protein biomass on natural gas using strain of methane-oxidising bacteria methylomonas koyamae b-3802d - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method of producing carotenoid-enriched protein biomass on natural gas for use in fodders for aquaculture, poultry and farm animals using a strain of methane-oxidising bacteria Methylomonas koyamae VKM B-3802D.

EFFECT: invention provides higher efficiency of cultivation of the new strain for production of protein biomass enriched with carotenoids, while maintaining high productivity.

1 cl, 3 ex

Description

The invention relates to biotechnology and the microbiological industry and is a strain of methane-oxidizing bacteria Methylomonas koyamae B-3802D, intended for producing carotenoid-enriched protein biomass using methane or natural gas for use in feed for aquaculture, poultry and farm animals.

Among the most promising areas of feed production technologies is the use of protein from unicellular microorganisms as alternative protein sources. Microorganisms of various taxonomic groups are used as unicellular protein producers. A special place in the production of bioprotein is occupied by methanotrophic bacteria - a metabolically unique group of prokaryotes capable of using methane (CH ₄ ) as the only source of carbon and energy. The use of methanotrophic bacteria as producers of protein biomass on methane or natural gas is effective, since on their basis it is possible to obtain a product containing up to 75% protein.

Strains of fast-growing thermotolerant methanotrophic bacteria belonging to the genus Methylococcus:Methylococcus capsulatus LBTI 028, VKPM V-13479 (RF patent No. 2728345);
Methylococcus capsulatusGBS-15, VKPM V-12549 (RF patent No. 2717991); Methylococcus capsulatusCONCEPT-8, VKM V-3289D (RF patent No. 2706074); Methylococcus capsulatus VS1, VKM B-3482D (RF patent No. 2765994); Methylococcus capsulatus BF19-07, VKPM V-13764 (RF patent No. 2745093). All of the above strains have a high growth rate of up to 0.25-0.28 h^-1, resistance to cultivation in the presence of methane homologues without reducing the growth rate and protein content in the biomass of 70-79%. High protein content is one of the main indicators when preparing balanced feeds. Other mandatory components of feed are biologically active additives of natural origin - carotenoids, which perform a number of physiologically important functions, such as stimulation of the immune system, provitamin activity, antioxidant and radioprotective properties, anti-carcinogenic effect, increasing tolerance to low oxygen concentrations, stimulating the growth process, accelerating puberty .

One of the obvious disadvantages of Methylococcus producer strains is their inability to produce carotenoids. Feed protein obtained from the use of methanotrophic producers of this genus requires additional enrichment with carotenoids synthesized by other microorganisms.

There is a known method for producing carotenoids using a pair of mutant strains of the filamentous fungus Blakeslea trispora 185B (+) and 185B (-) (RF patent No. 2053301). During joint submerged cultivation of strains in laboratory conditions for 114 hours, the level of accumulation of carotenoids averages 300 mg/100 ml of culture liquid. The disadvantages of the strain are the need for expensive components of the nutrient medium (soybean and corn flour, vegetable oil, thiamine, antioxidant, beta-ionone) and low growth rate. In addition, the cultivation of mycelial microorganisms in bioreactors is significantly difficult due to the high degree of aggregation of the biomass.

Another known method for the biosynthesis of carotenoids involves the use of the yeast strain Paffia rhodozyma K-16-43-34, VKPM Y-2982 (RF patent No. 2385925). The strain produces carotenoids in amounts up to 5.5 mg/g dry biomass. The yield of dry biomass is 80 g/l. In this case, the strain is grown in rich organic media with the addition of growth factors, and the synthesis of carotenoids is carried out under additional lighting conditions.

The closest analogue is the yeast strain Rhodotorula mucilaginosa 5-e, VKPM Y-4282 (RF patent No. 2662964), a producer of microbial protein and carotenoids. With strain productivity up to 30 g/l of protein, the content of carotenoids in the biomass is 650 μg/g. The protein content in biomass, however, does not exceed 30%. In addition to the limited nutritional value, the disadvantages of the strain include a strong cell wall, which requires additional processing of the resulting product. In addition, yeast is unable to use methane as a carbon source; using sugars as a substrate makes the process less economical.

The objective of the present invention is to find a new strain of methane-oxidizing bacteria for the biosynthesis of microbial protein enriched in carotenoids, expanding the range of biological raw materials for obtaining microbial protein mass.

The technical result of the claimed invention is to increase the efficiency of cultivating a new strain to obtain protein biomass enriched with carotenoids while maintaining high productivity.

The technical result was achieved by using the strain of methane-oxidizing bacteria Methylomonas koyamae MY1 , deposited in the All-Russian Collection of Microorganisms - VKM under registration number VKM B-3802D, as a producer to obtain microbial protein mass enriched with carotenoids. The strain designation assigned by the depositor is MY1.

Strain Methylomonas koyamae B-3802D was isolated from a sample of bottom sediments of an unnamed lake (Krasnodar region, Russia). The sample was selected from a horizon of surface sediments of 0-2 cm. The strain was isolated by cultivation on a mineral medium without the addition of growth factors and organic compounds at a temperature of 30°C and a pH of 6.5-7.0 in a methane-air atmosphere (1:3 ). The culture was purified from accompanying bacteria by the method of limiting dilutions of the enrichment culture, followed by sieving on a mineral agar medium and incubation in desiccators with 20% (v/v) methane in the gas phase.

The strain was identified using a comparative analysis of the 16S rRNA gene sequence and the complete genome, as well as based on the results of a study of physiological and biochemical characteristics. The systematic position of strain MY1 is as follows: phylum Pseudomonadota , class Gammaproteobacteria , order Methylococcales , family Methylococcaceae , genus Methylomonas , species Methylomonas koyamae . The work was carried out by the Federal Research Center of Biotechnology of the Russian Academy of Sciences and the Center for Shared Use "Bioengineering". The obtained sequences of the 16S rRNA gene and the genome of strain MY1 were deposited in NCBI under numbers OR234855 and CP133985.1, respectively.

Cultural and morphological characteristics of the strain indicating the environment:

When cultivated on an agar mineral medium with 20% (v/v) methane in the gas phase, the MY1 strain forms round, slimy, pigmented (bright yellow) colonies. When cultivated in a liquid mineral medium with methane in the gas phase, it forms yellow cell suspensions. The culture is represented by mobile rods 1.0–1.5 µm wide and 1.5–2.0 µm long with one polar flagellum. The type of cell wall structure is gram-negative. The cells contain stacks of densely packed intracytoplasmic membranes, characteristic of methanotrophic bacteria, located perpendicular to the cell wall.

Growth substrates are methane and methanol.

Assimilates as a source of nitrogen: potassium nitrate, ammonium sulfate, urea, yeast extract, atmospheric nitrogen.

Metals: increased demand for copper ions.

Does not require growth factors.

The culture is strictly aerobic.

Growth is observed in the temperature range 8-37°C (optimum 30°C) and pH 5.5-7.5 (optimum 6.5-7.0). The activity (productivity) of the strain is up to 3.5 kg×m ³ /hour. Method for determining the activity of the strain, indicating the method: continuous cultivation in a fermenter using a liquid mineral medium while maintaining the methane: air ratio = 1:3 in the gas-air mixture.

Method, conditions and composition of media for long-term storage of the strain: cryopreservation at - 80°C, 10% dimethyl sulfoxide (DMSO) solution is used as a cryoprotectant.

Method, conditions and composition of media for strain cultivation:

Option 1

Composition of the mineral medium for growing culture in flasks (g/l): KNO ₃ , 0.5; MgSO ₄ × 7H ₂ O, 0.5; CaCl ₂ × 2H ₂ O, 0.1; 100 mM phosphate buffer, pH 6.5, 1% (v/v); microelements solution 0.1% (v/v), containing (g/l): EDTA, 5; FeSO ₄ × 7H ₂ O, 2; ZnSO ₄ × 7H ₂ O, 0.1; MnCl ₂ × 4H ₂ O, 0.03; CoCl ₂ × 6H ₂ O, 0.2; CuSO ₄ × 5H ₂ O, 0.1; NiCl ₂ × 6H ₂ O, 0.02; Na ₂ MoO ₄ , 0.03. The methane content in the gas phase was adjusted to 30 vol. %. Cultivation was carried out on an incubator shaker at 150 rpm and a cultivation temperature of 30°C. pH value 6.5.

Option 2

Optimal conditions and composition of the medium for growing in a bioreactor:

H ₃ PO ₄ (87%) – 0.75 ml;

(NH ₄ ) ₂ SO ₄ – 0.9 g;

KCl – 0.25 g;

MgSO ₄ × 7H ₂ O – 0.25 g;

FeSO ₄ × 7H ₂ O – 0.02 g;

CuSO ₄ ×5H ₂ O – 0.016 g;

MnSO ₄ × 5H ₂ O – 0.021 g;

NiSO ₄ × 7H ₂ O – 0.001 g;

ZnSO ₄ × 7H ₂ O – 0.003 g;

H ₃ VO ₃ –0.008 mg;

CoSO ₄ ×7H ₂ O - 0.0004 mg;

Na ₂ MoO ₄ × 2H ₂ O - 0.0004 mg.

Genetic characteristics of the strain: a) no mutations, deletions, or inversions; b) phage resistant; c) no plasmids were detected; d) prophages were not detected. Information about the safety of using the strain: a) the strain is not genetically modified and does not contain genes of other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; b) the strain is not zoopathogenic or phytopathogenic; does not pose a danger for any other reason.

The claimed strain is characterized by:

- sustainable productive growth on mineral media with methane as a growth substrate, over a wide range of pH and temperature, with a high specific growth rate of up to 0.29 h ^-1 ;

- ability to synthesize carotenoids (lycopene derivatives);

- lack of virulence, toxigenicity, toxicity and harmlessness.

Cultivation is carried out mainly in a continuous manner, in bioreactors with mineral media and methane (or natural gas) as a substrate.

The invention is illustrated by the following examples.

Example 1.

Cultivation of the Methylomonas koyamae MY1 strain was carried out in batch mode in heat-resistant glass flasks with a volume of 0.75 l with a filling factor of 0.3 on a thermostatic shaker at 30°C. Cultivation was carried out for 48 hours. At the end of cultivation, the concentration of dry substances was 0.8 g. The carotenoid content was 86 μg/g dry weight. The resulting culture was used as seed material for the subsequent cultivation of bacteria in an automated fermentation complex with a volume of 1.5 l (working volume 1.0 l).

Example 2.

The culture of Methylomonas koyamae MY1 was grown in a continuous mode in a fermenter with a volume of 1.5 l (working volume - 1.0 l) with a continuous supply of a mineral medium containing the following components (per 1 l of medium):

H ₃ PO ₄ (87%) – 0.75 ml;

(NH ₄ ) ₂ SO ₄ – 0.9 g;

KCl – 0.25 g;

MgSO ₄ × 7H ₂ O – 0.25 g;

FeSO ₄ ×7H ₂ O - 0.02 g;

CuSO ₄ ×5H ₂ O – 0.016 g;

MnSO ₄ ×5H ₂ O – 0.021 g;

ZnSO ₄ × 7H ₂ O – 0.003 g;

H ₃ VO ₃ - 0.008 mg;

CoSO ₄ ×7H ₂ O - 0.0004 mg;

NiSO ₄ ×7H2O – 0.001 g;

Na ₂ MoO ₄ × 2H ₂ O - 0.0004 mg.

The growing process was carried out under aseptic conditions, at a flow rate of 0.22 h ^-1 . The pH value was adjusted by titration with a 1% ammonia water solution, which also served as an additional source of nitrogen. The process temperature was regulated by supplying cooling water to the heat exchanger of the apparatus.

The consumption of natural gas and air per 1 liter of culture medium was 6 and 18 l/hour, respectively.

When maintaining this cultivation regime for a long time, the concentration of biomass in the fermenter was 3.2 g/l. Protein content - 68.25%, carotenoids - 48.55 µg/g dry biomass.

Example 3.

The culture of Methylomonas koyamae MY1 was grown in a continuous mode in a fermenter with a volume of 1.5 l (working volume - 1.0 l) with a continuous supply of a mineral medium containing the following components (per 1 l of medium):

H ₃ PO ₄ (87%) – 0.75 ml;

(NH ₄ ) ₂ SO ₄ – 0.9 g;

Urea – 0.1 g;

KCl – 0.25 g;

MgSO ₄ × 7H ₂ O – 0.25 g;

FeSO ₄ ×7H ₂ O - 0.02 g;

CuSO ₄ ×5H ₂ O - 0.16 g;

MnSO ₄ ×5H ₂ O - 0.21 g;

NiSO ₄ ×7H2O – 0.001 g;

ZnSO ₄ × 7H ₂ O – 0.003 g;

H ₃ VO ₃ - 0.012-0.008 mg;

CoSO ₄ ×7H ₂ O - 0.0004 mg;

Na ₂ MoO ₄ × 2H ₂ O - 0.0004 mg.

The growing process was carried out under aseptic conditions, at a flow rate of 0.22 h ^-1 . The pH value was adjusted by titration with a 1% ammonia water solution, which also served as an additional source of nitrogen. To stimulate the biosynthesis of carotenoids, the inclusion of urea in the medium was used. The process temperature was regulated by supplying cooling water to the heat exchanger of the apparatus.

The consumption of natural gas and air per 1 liter of culture medium was 6 and 18 l/hour, respectively.

When maintaining a continuous cultivation regime for 30 days, the biomass concentration in the fermenter was 3.5 g/l. Protein content - 75.82%, carotenoids - 111.93 µg/g dry weight.

The data obtained indicate that continuous cultivation of the strain Methylomonas koyamaeMY1 in a mineral environment of the specified composition allows to achieve high productivity of the process (biomass yield per unit of time). Strain Ability Methylomonas koyamaeMY1 producing carotenoids allows you to obtain protein biomass enriched with carotenoids.

Claims (2)

1. A method for producing carotenoid-enriched protein biomass using natural gas using a strain of methane-oxidizing bacteria Methylomonas koyamae B-3802D when cultivated in a fermenter with a geometric volume of 1.5 l in a continuous mode under aseptic conditions with a continuous supply of a mineral medium at a dilution rate of 0.22 h ^-1 , titration with a 1% ammonia water solution at a flow rate of natural gas and air per 1 liter of culture in a ratio of 6:18 l/h, ensuring a biomass concentration of 3.2 g/l, protein content of 68.3%, carotenoids - 48 .6 µg/g dry biomass. 2. A method for producing carotenoid-enriched protein biomass using natural gas using the strain of methane-oxidizing bacteria Methylomonas koyamae B-3802D according to claim 1, characterized in that urea is additionally added to the cultivation medium in an amount of 0.1 g/l, which ensures the production biomass with a concentration of 3.5 g/l, protein content - 75.82%, carotenoids - 111.93 μg/g dry weight.