RU2103358C1 - Strain of bacterium arthrobacter terregens vsb-570 - a producer of protein and biopreparation for petroleum product treatment - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention proposes mesophilic strain of bacterium Arthrobacter terregens VSB-570-VKPM-S-1180 for soil treatment polluted with petroleum products. Strain grows actively on different waste: bran, nonconditioned grain, plant hydrolyzates, from meat and fat and oil industry. Strain produces crude protein at amount 15-20% more as compared with yeast C. maltosa (salmonicola), C. tropicalis, C. scotti, C. utilis. Strain can grow on crude oil and its dried biomass can be used as a biopreparation for soil treatment polluted with petroleum products. Also, biomass dried under severe conditions can be used for technical aims. EFFECT: strain indicated above, enhanced effectiveness of strain.

Description

 The present invention relates to biotechnology and for the production of feed and technical protein from agricultural and industrial wastes when using the protein of the high-protein bacterial strain Arihrobacter terregens VSB-570-VKPM-S-1180 as a producer.

 It is known that large-scale plants for the production of protein both in plant hydrolysis and in hydrocarbons of oil as yeast producer used yeast species: C.tropicalis, C.scottii, C.utilis, which provide a crude protein content in the finished product of 46-56% on hydrolysates, yeast p. Candida 58-63% on paraffins [1,2]. A disadvantage of the invention of Candida yeast strains is the relatively low crude protein content in the finished product, the weak growth of hydrolysis yeast from the waste products of the meat, fat industry and crude oil.

 The search for additional carbon sources to obtain feed protein for animal husbandry or technical protein for reinforced concrete structures or asphalt will allow its deficit in the country. Various agricultural wastes (bran, substandard grains, hydrolysates of plant materials with a high content of such inhibitors as furfural, volatile acids), wastes of the meat and fat industry allow us to solve several important economic problems: on the one hand, expanding the cheap range of carbon power sources for getting scarce protein, on the other - environmental, reducing the burden on treatment facilities of enterprises.

 The technical result of the invention is to improve the quality of products by protein content and yield of finished products on various agricultural and industrial wastes.

 This technical result is achieved by applying a high-speed and high-protein non-pathogenic culture of Arthrobacter terregens VSB-570-VKPM-S-1180.

 The bacterial culture of Arthrobacter terregens VSB-570-VKPM-S-1180 was isolated as concomitant microflora during technological cultivation in a continuous process of yeast of the R. Candida maltosa in crude Mangyshlak oil. The strain was identified by the identifier Bergi (1980) as Arthrobacter terregens VSB-570 and deposited in the All-Russian collection of industrial microorganisms of the Institute VNIIgenetika under the number VKPM-S-1180, in the collection of the State Research Institute of Synthesis protein is stored under N VSB-570.

 Morphological signs: old cultures consist mainly of coccoid cells, single in pairs or in clusters, when replanted on fresh medium, growth occurs by increasing coccoid cells, after which one or sometimes both ends of the cell lengthens, resulting in the formation of sticks. Propagated by division. The rods are motionless; they do not form endospores. Cell size: cocci 0.63 x 0.63 microns, 0.63 x 0.77 microns and rods 1.25 x 0.91 microns to 1.75 x 2.1 microns. Colonies on meat-peptone agar (MPA) are small, from punctate to 2 mm, round, the edge is smooth, the surface is smooth, slightly shiny, raised to the center, grayish in color. On the BCH, growth is in the form of a very thin film over the entire surface, the haze is weak, the precipitate is medium grayish-white in color, Gram stain is positive.

Physiological signs: aerob, growth maximum at a temperature of 28-34 ^o C, pH 6.8-7.2. When sowing with a prick in a column, meat-peptone gelatin (MF), growth is observed on the surface in the form of a hat, the prick is clear, threadlike. Gelatin liquefaction is not observed, milk is restored by litmus, milk clotting is not observed. The content of G + C in DNA is in the range of 60-72 mol.%. Of the carbon assimilates; glucose, mannose, sucrose, maltose, galactose, xylose, 1-arabinose, raffinose. It actively develops on a mineral medium with an autolysate using plant waste (rice, corn husk, tombs, bran, defective grain), wide-fraction paraffins, oil, petroleum distillate, ethyl alcohol, and waste from the meat and fat industries as the sole carbon source.

 The invention is illustrated by the following examples.

Example 1. Periodic cultivation was carried out in rocking flasks on mineral medium N 10 of the following composition: (NH ₄ ) ₂ SO ₄ 3 g / l, KH ₂ PO ₄ 6.5 g / l, MgSO ₄ 0.7 g / l, and trace elements FeSO ₄ 12.5 mg / l, ZnSO ₄ 12.5 mg / l, MnSO ₄ 12.5 mg / l, CuSO ₄ 3 mg / l, at a temperature of 30-32 ^o C, pH 7.0. Waste from the meat and fat industry at a concentration of 20 vol.% Was used as a carbon source. % of the following composition: fat content of 2 g / l (lipoprotein substances), suspended solids of 3 g / l, COD 4000 mg / l, BOD ₅ 1500 mg / l. Growing for 48 hours on a circular rocking chair with 220 rpm. Inoculation was carried out at an optical density of 0.1 units with a one-day culture washed with MPA. As a result of fermentation, 3.9 g of ASB / L were obtained, a reduction in COD of 90-100 mg / L, BOD ₅ 10 mg / L.

Dried at 80 ^o C in an oven, the biomass is characterized by the content,%: crude protein 67, true protein 47-48, lipids 3, ash 7, moisture 8.3.

Example 2. Periodic cultivation was carried out in rocking flasks on the mineral medium specified in example 1, the hydrolyzate of the Krasnodar chemical plant of the following composition was used as a carbon source: reducing substances 3.5%, nitrogen 4.74 g / l, phosphorus 120 mg / l, furfural 0.01%; volatile acids 0.34%; solids 1.61%; pH 6.85. The cultivation temperature was 32-34 ^o C, inoculum was introduced in the form of a one-day suspension of the bacterial strain Arthrobacter terregens VSB-570-VKPM-S-1180 at an optical density of 0.1 units.

 As a result of fermentation, a biomass concentration of 4.85 gASB / L of the following quality was obtained: crude protein content of 68%, true protein of 49%, lipids of 8.3%. The strain Arthrobacter terregens VSB-570 is resistant to inhibitors such as furfural and volatile acids.

Example 3. The bacterial strain Arthrobacter terregens VSB-570-VKPM-S-1180 was grown under laboratory conditions in a batch cultivation mode at a temperature of 30-32 ^o C, pH 6.8-7.0, bran weight 5 was used as a carbon source .% or substandard grain 5 wt.%, which are characterized by the content of lightly hydrolyzed polysaccharides 51.9 g / l, hardly hydrolyzed polysaccharides 15 g / l, nitrogen total 2.7 g / l, fiber 1.1%, starch (linked monosaccharide) 4.4%, ash 5.4%. In addition to a carbon source, 100 mg of water additionally contains 100 mg of (NH ₄ ) ₂ SO ₄ , 200 mg of K ₂ HPO ₄ . Inoculation was carried out at an optical density of 0.1 units with a one-day culture of bacteria Arthrobacter terreegens VSB-570-VKPM-S-1180. As a result of fermentation (36 h), the biomass was separated by centrifugation, dried at 70 ^o C in an oven, which is characterized by a crude protein content of 45-48%. The bacterial strain provided a deeper utilization of reducing substances up to 0.06 g / l, carbohydrates up to 0.95 g / l. As a result of a periodic experiment in laboratory and laboratory conditions, it was found that the strain Authrobacter terregens VSB-570-VKPM-S-1180 is actively growing on bran and substandard grain.

Example 4. The experiment was conducted in laboratory conditions in a typical fermenter with a volume of 10 l on a nutrient medium of the following composition, in g / l:
Phosphoric acid (70%) - -2.5
Potassium Chloride - 1.14
Magnesium Sulfate - 0.55
Sulphate of iron - 0,046
Zinc sulfate - 0,033
Copper sulfate - 0.004
Manganese Sulfate - 0.026
Nitrogen in the amount of 2.45 g / l is supplied in the form of a 6% aqueous solution of ammonia, while using it to control the pH of the medium in the range of 6.8-7.0.

The fermentation temperature is 30-32 ^o C, the pH of the medium is 6.8-7.0. Crude oil was used as a carbon source at a concentration of 1 vol.% One-day seed was introduced from rocking flasks at a concentration of 0.4 g / l of pressed biomass. When the flow rate of the medium D = 0.25 h ^{-1 the} fermenter productivity reached 3.6 kg / m ³ / h, the yield from the content of n-alkanes in the feed is 97%.

 The resulting biomass is characterized by the content, in%: crude protein 68, true protein 51.2, lipids 6.0, carbohydrates 5.38, ash 11.7, moisture 4.3, nucleic acids 9.0, sulfur content of amino acids 2.3 to the DIA. The biomass obtained from oil during soft drying can be used as a biological product for cleaning oil products and for technical purposes.

 Thus, the proposed bacterial strain Arthrobacter terregens SVB-570-VKPM-S-1180 provides additional feed for livestock from meat and fat industry, agriculture: bran, substandard grain, hydrolysates of plant materials containing inhibitors such as furfural, volatile acids.

 When cultivating the proposed bacterial strain on crude oil, it is possible to obtain a biological product for soft cleaning from petroleum products and technical protein by soft drying.

Claims (1)

 The bacterial strain Arthrobacter terregens VSB-570-VKPM-S-1180 is a protein and biological product producer for purification of oil products.