The study of the effect of nutrient medium composition for azotobacter Chroococcum on the rheological and physico-mechanical properties of biopolymer

Introduction. In recent years when intense environmental pollution is increasing, traditional synthetic polymers based on plastics have been replaced by so-called compostable plastics (biodegradable plastics). Compostable plastics allows polymer materials to decompose in natural conditions under the action of external and intracellular enzymes of microorganisms to practically harmless compounds without harming the environment.

Reproducible natural polymers, components of agricultural or wild plants (starch, cellulose, lignin), petrochemical products, or combined technologies can serve as a raw material basis for the production of modern biopolymers [2].

The importance of polymers in modern society is difficult to underestimate. The main directions of the world economy are closely related to the production and consumption of polymers. In recent years, there has been a positive trend in the development of this area, as a result, the problem of waste disposal after the expiration of the operational life of the materials themselves and products made from them is acute. The polymers produced are highly valued because of their high resistance to micro-organisms. At the same time, this becomes a problem, because synthetic polymers in the environment are immune to external factors and do not decompose in natural conditions. In this regard, researchers are increasingly interested in the task of making synthetic polymer systems biodegradable, which would retain their consumer properties during the service life, and after it synthetic polymer systems will transform physically and biochemically into harmless components. Plastics, polymer materials produced from oil are widely used all over the world. With increasing demand, recycling of plastic waste has become a major global problem. In this regard, the development of new plastics, polymer materials that can be decomposed by microorganisms in soil and sea water is very widespread [3].

Objects and methods of research. The object of research is a biopolymer based on Azotobacter chroococcum. The strain of the microorganism Azotobacter chroococcum (B-5787) D–08 was obtained from the fund of the all - Russian collection of industrial microorganisms of the Kurchatov Institute-Gosniigenetika. Azotobacter chroococcum is a gram-negative bacterium discovered in 1901 by Martinus Beyerink, a well-known virologist. Beyerink isolated from the soil an aerobic non-spore-forming bacterium that fixes molecular nitrogen, and named it Azotobacter chroococcum (the generic name reflects the ability of the bacterium to fix nitrogen, in the species - the ability to synthesize brown pigment-chroo and form coccoid cells-coccum). To fix nitrogen, Azotobacter chroococcum produces three enzymes (catalase, peroxidase, and superoxide dismutase) to «neutralize» reactive oxygen species. Azotobacter is a typical representative of free-living microorganisms. Free-living organisms are all those microorganisms that live in the soil, regardless of whether the plant develops nearby or not.

Biodegradable polymer materials retain their properties almost unchanged during the service life, at the end of which they undergo accelerated physical, chemical and biological transformations in the natural environment, easily being included in the metabolism of polysystems. For the disposal of polymer materials, it is not necessary to allocate an additional area for landfills, and decomposition products are not able to have a negative impact on the environment. Modern industry is actively introducing biopolymers as an affordable and biodegradable packaging material. At this stage of biopolymer research, an important task is to develop the necessary regulatory standards governing the testing methodology and quantitative parameters of the biodegradation of recycled residues.

The study of vapor permeability of laboratory samples was carried out in accordance with «GOST 21472 Materials, gravimetric method for determining vapor permeability».

First, the material selected in accordance with the requirements were flat, clean, without mechanical damage. Samples for testing were cut out in the form of a disk according to a template, with a diameter of 5 centimeters. Then conditionally designated by numbers. Those samples of the material were placed in laboratory conditions for 4 hours before testing.

Prepared and covered with a lid, the device for determining vapor permeability was placed for 15 minutes in a room to equalize the temperature, then we weighed it. With tongs, the device was transferred to the camera and the cover was removed. After 24 hours, the device was closed with a lid and forceps were taken out of the chamber, cooled to room temperature and weighed with an error of no more than 0.0001 g. The test results were plotted on a graph of the time-dependent mass change.

The laboratory samples were tested for tensile strength in accordance with GOST 14236-81 «polymer films. Method of tensile testing». For testing, samples were taken with smooth edges, without notches. Rectangular shape reaches with a width of 20mm and a length of 150mm. The samples are fixed in the clamps of the testing machine. They are evenly tightened so that the sample does not slip during the test, but the sample does not collapse at the point of attachment. The samples are then conditioned for 16 hours at 23°C and 50% relative humidity. Before testing, the central part of the sample is marked with marks that limit the calculated length. The thickness and width of the samples are measured in three places, in the middle of the sample and at a distance of 5 mm from the edges of the marks.

Laboratory samples were tested for water absorption in accordance with GOST 46502014 (ISO 62:2008) «Plastics. Methods for determining water absorption». The test samples are immersed in distilled water at a temperature of 23°C or boiling distilled water or kept in an atmosphere with a relative humidity of 50% at a given temperature for a set period of time. The mass of water absorbed by each test sample is determined or calculated from the difference between the mass of the sample before and after the test, expressed as a percentage of the initial mass. If necessary, the mass of water lost by the test sample after drying can be determined. Before the test, rectangular samples were selected. 1 mm thick, 100 mm long. Then the samples must be thoroughly dried at temperature of 50°C.

The density of laboratory samples was studied in accordance with GOST 15139-69 (ISO 1183) «Methods for determining density». Samples should be smooth and free of voids and cracks to reduce the possibility of air bubbles being trapped or incorporated. The volume of the material is 1 cm3 and the weight is 180g. Samples are conditioned before testing and tested in accordance with GOST 12423-66 under the standard control atmosphere. The sample volume is determined as follows: the correct form is calculated according to the results of measurements, inaccurate, or difficult to be measured forms the volume of the displaced fluid. To do this, the device, which is in a vertical position with a narrow neck up, is filled with liquid to the lower division of the scale or slightly higher, closed with a cork and turned 360°, then the liquid level is counted. Then the device is turned over so that the large stopper is at the top, after that the weighted sample with a volume of 5 cm3 is inserted into the device, tightly closed with a stopper and, turning it over, brought to its original position.

The new liquid level is counted, making sure that there are no air bubbles sticking to the sample. The volume of the sample is determined by the difference between the liquid levels.

The study of biodegradation from the laboratory samples was carried out in accordance with GOST 9.060-75 «Unified system of protection against corrosion and aging (ESZKS). Cloths. Method of laboratory tests for resistance to microbiological destruction» [1]. A 25×300 mm spot sample is taken from each selected piece. For testing, we prepared a mixture (hereinafter referred to as soil) of sand, horse manure and garden land, taken in equal amounts by weight. The soil prepared before testing must be kept for at least two months at (20±5)°C. Before testing, the soil is sifted through a sieve and water is added to form a homogeneous slurry. The ratio of land and water by weight is 2: 1. the Test strip is placed on a flat glass surface. In the center of the test strip, apply a stencil so that the hole in the stencil coincides with the edges of the strip.

The prepared soil is applied with a spatula over the entire area of the stencil opening. After applying the soil, the ends of the test strip are connected in a loop so that the soil is on the outer surface. The ends of the resulting loop are fixed with a clothespin on a glass stick placed between two tripods. A wet asbestos plug is inserted inside the loop, which serves to maintain the specified humidity of the test strip of fabric and thereby keeps the soil on the outer surface of the loop. Then glass sticks with test strips are placed in a vessel, which is pre-filled with water 25 mm high. The vessel is closed with a lid and placed in a thermostat. Tests in the thermostat are carried out at 28°C. The test duration is 10 days. After testing, the test strips are carefully cleaned from the soil, washed with water until it is completely removed, treated with an aqueous solution of formalin at the concentration of 1-2 g / DM (40%) for 15 minutes and air-dried. The dried test strips and the original test strips are kept in atmospheric conditions.

The stability of the initial films in water was determined in accordance with GOST 12020-72.

The essence of the method is to determine the change in the mass of samples after exposure for 24 hours in a model medium (distilled water).

For testing, samples are cut out in the form of a disk with a diameter of 50 mm. Before testing, the samples are conditioned for 88 hours at 23°C and 50% relative humidity without the influence of light. Then each sample is weighed in a glass closed vessel (bux) and its linear dimensions are measured. The thickness of the sample is measured at four points. The samples are placed in a vessel with a chemical reagent heated to the test temperature. The samples are placed in a vessel so that they are completely immersed in the chemical reagent (the samples should not come into contact with each other and with the walls of the vessels) and kept at a temperature of 20° C [1, 5].

Discussion of results. In the course of our research, a bio-binding agent based on the exopolysaccharide Azotobacter chroococcum strain (B–5787) D-08 was obtained. The culture was grown for 72 hours at 28° C.

In order to obtain an inoculate, Azotobacter chroococcum strain (B-5787) D–08 was grown on a liquid sugar-containing medium of the following composition: g / l: K2HPO4-0.8; KH2PO4-0.2; CaSO4 * 7H2O – 0.2; Mdso4 * 7H2O – 0.2; Na2MnO4 – 0.05; FeCl 3 – 0.05; yeast extract – 0.5; sucrose – 20.0.

Azotobacter chroococcum was cultured in a temperature-controlled shaker for 72 hours at 250 rpm and the temperature of 28° C. The resulting inoculate was sown in nutrient media, which included food production waste-molasses, post-alcohol bard and whey (in a ratio of 1:1:1) with the addition of lignocellulose fillers in a ratio of 1:3, 1:6 to the nutrient medium. Further cultivation was carried out for 72 hours (3 days) at 250 rpm and the temperature of 28° C.

It is known that the growth of Azotobacter bacteria requires components such as carbohydrates, alcohols, organic acids, and minerals in the form of phosphoric and calcium salts. The food production waste we use contains substances necessary for the growth of the microorganism, as well as for the formation and accumulation of Levan polysaccharide.

We noted that the type of lignocellulose filler in the culture medium did not negatively affect the growth and development of the bacterium Azotobacter chroococcum strain (B-5787) D–08.

On the third day, the culture liquid obtained by us had a thick and viscous consistency.

The environment used in the experiment contained a mixture of beet pulp and straw in the ratio of filler 1:3 in relation to the nutrient medium.

At the stage of biopolymer preparation, fillers were added to the suspension, and changes in rheological and physico-mechanical properties were monitored.

In this work, we used the vpj-1 viscometer, designed to determine the kinematic viscosity of liquid media. Viscometers of this type allow you to measure the viscosity of liquids at positive and negative temperatures. This viscometer is made of chemical laboratory glass in accordance with the requirements of GOST 21400-75.

When studying the physical and mechanical properties of a laboratory sample of a biocomposite material made of straw, sugar beet pulp and Levan polysaccharide, the strength limits were established. The density is 1.17 g / cm3, the tensile strength in the longitudinal direction is 22 MPa, the strength in the transverse direction is 19 MPa, and the elongation at break is 6%. The material is not suitable for vacuum packaging: the vapor permeability was 150 g / m2sut., water absorption at room temperature -23%.

Biodegradable substance based on natural polymers, has a short biodegradation time of up to 45 days, is environmentally safe and does not require large production costs. The product fully meets the requirements of GOST for biodegradable materials for industrial use [4, 6].

Conclusions. Having conducted a comprehensive study of the literature and based on our own research and experiments, we can make the following conclusions from this work:

Our experiments have shown that the use of beet pulp, straw, and a Levan-containing bio-binder makes it possible to obtain a material which forms the conditions for a larger number of adhesive layers due to the adhesive properties of Levan and substances that make up beet pulp. This is caused by the presence of pectin substances and binding glycans (vegetable cellulose) in beet pulp. Such a dense structure of the material can serve as a prerequisite for high physical and mechanical properties. Developed an optimal component composition, a biodegradable material having a ratio in the composition of straw and beet pulp (the ratio of lignocellulosic filler to a nutrient medium 1/3), and selected technological parameters cultivation time: 72 hours (3 days) at 250 rpm and 28° C, and the parameters of the evaporation and drying in a drying cabinet at 50° C. The dried mass was loaded into a mold and subjected to hot pressing at a temperature of 140° C and a pressure of 19.6 MPa (15 t) for 5 minutes. The physical, mechanical and technical properties of the biodegradable material were studied. The density is 1.17 g / cm3, tensile strength - from 22 MPa, elongation at break from 9%, vapor permeability 150 g /m2, water absorption at the room temperature-23%, withstand ambient temperature up to 70°C, moisture 75%, when using products, contact with alkalis and acids is not allowed.

Thus, it can be argued that the use of biopolymers based on Azotobacter chroococcum producing Levan polysaccharide is more appropriate and cost-effective in comparison with synthetic polymers available on the market. The resulting biocomposite polymer material is recommended to be used as a material for packaging dry, bulk and solid products.