RU2802498C1 - Pseudomonas helmanticensis polyhydroxyalkanoate producer resistant to sodium dodecyl sulfate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Pseudomonas helmanticensis P1 strain, which has the ability to accumulate polyhydroxyalkanoate in the presence of sodium dodecyl sulfate, is deposited in the Network Bioresource Collection in the field of genetic technologies for agriculture under the number RCAM05967.

EFFECT: invention provides an extension of the arsenal of methods for producing polyhydroxyalkanoate in non-sterile media containing sodium dodecyl sulfate.

1 cl, 2 ex

Description

The invention relates to the field of biotechnology and is associated with the isolation of a sodium dodecyl sulfate (SDS)-resistant polyhydroxyalkanoate (PHA) producing strain.

PHAs are polyesters produced biosynthetically by bacteria Bacillus , Cupriavidus , Pseudomonas , etc. (Zinn, Witholt, Egli, Advanced Drug Delivery Reviews, 2001, V. 53, N.1, P. 5-21) They have many applications both as packaging materials and in the production of medical products. PHAs have a number of advantages over traditional polymers obtained by petroleum refining. In particular, they quickly decompose both in natural ecosystems and in the human body without the formation of toxic products (Bonartsev, Bonartseva, Reshetov, Kirpichnikov, Shaitan, Acta Naturae, 2019, V. 11, N. 2, P. 4-16) .

However, the high cost of producing PHAs significantly limits their use. High production costs are associated, among other things, with high energy costs. An integral stage of most PHA production processes is steam sterilization of media. To obtain several kilograms of polymer, at least a ton of sterile medium is required. It is obvious that sterilizing culture media on an industrial scale consumes a significant amount of electricity.

Steam sterilization can be avoided by using selective media that can only be metabolized by the PHA producer. The selectivity of the medium can be increased by adding to it any antimicrobial agent to which the producer has tolerance. Such an agent may be a surfactant such as SDS. SDS prevents the growth of most microorganisms (Johnston, Simons, Lambert, Journal of Applied Microbiology, 2000, V. 88, N. 2, P. 237-242), however, some polyhydroxyalkanoate-producing Pseudomonas bacteria are resistant to its action (Zubkov, Nepomnyshchiy, Kondratyev , Sorokoumov, Sivak, Ramsay, Shishlyannikov, Journal of Microbiology, 2021, V. 59, N. 12, P. 1104 – 1111). Thus, the addition of SDS to the medium allows cultivation under non-axenic conditions and thereby reduces the cost of PHA production.

The purpose of the invention is to obtain an SDS-resistant PHA producer capable of accumulating biomass and polymer in the presence of toxic (up to several tenths of g/l) concentrations of SDS.

The Pseudomonas helmanticensis P1 strain was isolated from a sample of contaminated soil collected within the city of Gatchina, Leningrad Region. To isolate the strain, a suspension of the soil sample was applied to an agar medium containing 5 g/L SDS as the sole carbon source and cultivated at 28°C.

On the surface of agar media (MPA) after 24 hours at 28°C, P. helmanticensis P1 forms beige, convex, smooth colonies with smooth edges up to 3 mm in diameter. Gram-negative bacteria, non-spore-bearing, rod-shaped cells measuring 0.5-1.0×1.5-2.5 µm, forming aggregates surrounded by a protective matrix in the presence of SDS.

The optimal growth temperature is 27-31°C, the optimal pH range is 5.5-7.0. Does not grow at temperatures below 4°C and above 37°C. Capable of growth in the presence of up to 5 g/l SDS. Does not hydrolyze starch. Does not reduce nitrates.

According to the nucleotide sequence of the variable region V3-V4 of the 16S rRNA gene, it is closest to the species P. helmanticensis (degree of relationship 99%). Stored on slanted agar media (SMA) at 4°C for two months and at -80°C in the presence of 20% glycerol for two years.

There is no information about the pathogenicity of P. helmanticensis in the scientific literature.

The use of the P. helmanticensis P1 strain is illustrated by the following examples.

Example 1. Preparation of polyhydroxyalkanote using P. helmanticensis P1 in a medium containing glycerol and SDS

P. helmanticensis P1 was cultivated in a liquid medium containing 10 g/l glycerol, 0.5 g/l NH ₄ Cl, 0.5 g/l (NH ₄ ) ₂ SO ₄ , 3 g/l Na ₂ HPO ₄ 12H ₂ O, 2 g/l KH ₂ PO ₄ ·H ₂ O, 0.01 g/l MgSO ₄ and 0.5 g/l SDS, in 750 ml conical flasks. The volume of cultivation medium in the flask was 90 ml, the volume of added inoculum was 10 ml. The culture medium was not pre-sterilized. Cultivation was carried out in an incubator shaker at 28°C and a stirring speed of 250 rpm for 96 hours. At the end of cultivation, the biomass was separated by centrifugation (10 min at 6800 × g), dried at 70°C to constant weight and crushed in a mortar.

Extraction of PHA from dry biomass was carried out according to the method (Rebocho, Pereira, Freitas, Neves, Alves, Sevrin, Grandfils, Reis, Applied Food Biotechnology, 2019, V. 6, N. 1, P. 71-82). 1 g of dry biomass was extracted three times with 50 ml of chloroform at 65°C for 6 h. The extracts were combined and concentrated in vacuo to a volume of 1 ml. The polymer was precipitated with 10 mL of cold (4°C) ethanol, separated by centrifugation (10 min at 6800 × g) and dried at room temperature to constant weight. From 1 g of biomass, 250 mg of PHA was isolated, which corresponds to the degree of conversion of glycerol into the product of 6.5%.

Example 2: Preparation of polyhydroxyalkanote using P. helmanticensis P1 in a medium containing sodium octanoate and SDS

P. helmanticensis P1 was cultivated in a liquid medium containing 10 g/l sodium octanoate, 0.5 g/l NH ₄ Cl, 0.5 g/l (NH ₄ ) ₂ SO ₄ , 3 g/l Na ₂ HPO ₄ 12H ₂ O, 2 g/l KH ₂ PO ₄ ·H ₂ O, 0.01 g/l MgSO ₄ and 0.5 g/l SDS, in 750 ml conical flasks. The volume of cultivation medium in the flask was 90 ml, the volume of added inoculum was 10 ml. The culture medium was not pre-sterilized. Cultivation was carried out in an incubator shaker at 28°C and a stirring speed of 250 rpm for 96 hours. At the end of cultivation, the biomass was separated by centrifugation (10 min at 6800 × g), dried at 70°C to constant weight and crushed in a mortar.

Extraction of PHA from dry biomass was carried out according to the method (Rebocho, Pereira, Freitas, Neves, Alves, Sevrin, Grandfils, Reis, Applied Food Biotechnology, 2019, V. 6, N. 1, P. 71-82). 1 g of dry biomass was extracted three times with 50 ml of chloroform at 65°C for 6 h. The extracts were combined and concentrated in vacuo to a volume of 1 ml. The polymer was precipitated with 10 mL of cold (4°C) ethanol, separated by centrifugation (10 min at 6800 × g) and dried at room temperature to constant weight. Then the polymer was redissolved in 1 ml of chloroform and precipitated with 10 ml of cold (4°C) ethanol. Reprecipitation was repeated twice to remove the remaining octanoic acid. From 1 g of biomass, 340 mg of dry PHA was isolated, which corresponds to the degree of conversion of sodium octanoate into the product of 8.5%.

Thus, P. helmanticensis P1 strain can be used to produce PHA in nonsterile media containing SDS. The addition of SDS inhibits the growth of foreign microorganisms, which allows cultivation under non-axenic conditions. The absence of a stage of steam sterilization of media reduces the cost of the finished PHA by reducing energy consumption.

Claims (1)

The bacterial strain Pseudomonas helmanticensis P1, deposited in the Network Bioresource Collection in the field of genetic technologies for agriculture (RCAM) under the number RCAM05967, is a polyhydroxyalkanoate producer, resistant to up to 5 g/l sodium dodecyl sulfate.