Bacillus Subtilis SK01 and its Application in Plastic Degradation

Abstract

Bacillus subtilis SK01 and its application in plastic degradation. Bacillus subtilis SK01 collected by China Center for Type Culture Collection (CCTCC), CCTCC NO: M2020812 is used to prepare a degradable plastic that can protect Bacillus subtilis SK01 from the damage of high temperature and reduce the difference with polyene plastics in specific gravity. The plastic may be stored for more than 1 year to ensure that the microbial enzymes produced by Bacillus subtilis SK01 can achieve 49.11% of total degradation of organic solids after 153 days in an environment for composting or soil burial, and can be slowly degraded by sunlight (photodegradation) or immersion in water. Under an environment other than that mentioned above, the finished products of polyene plastics added with Bacillus subtilis SK01 are consistent with the non-degradable plastics in shelf life, satisfying the demand of a variety of polyene plastics.

Claims

1. Bacillus subtilis SK01, collected by China Center for Type Culture Collection CCTCC NO: M2020812.

2. A microparticle capsule of Bacillus subtilis, wherein a coat for Bacillus subtilis SK01 according to the claim 1, which consists of lactic acid or chitosan and methyl silicone oil.

3. The microparticle capsule of Bacillus subtilis according to the claim 2, wherein the dosage of each component is: 49˜53% of Bacillus subtilis SK01, 45˜47% of lactic acid or chitosan, and 1˜4% of methyl silicone oil (by weight percentage of dry solid form).

4. A biodegradable plastic masterbatch produced by banburying and granulation of the Bacillus subtilis microparticle capsule, polyolefin, calcium carbonate, dispersant and zinc stearate according to the claim 2.

5. A biodegradable plastic masterbatch produced by banburying and granulation of the Bacillus subtilis microparticle capsule, polyolefin, calcium carbonate, dispersant and zinc stearate according to the claim 3.

6. The biodegradable plastic masterbatch according to the claim 4, wherein the dosage of each component is: 0.3˜1% of Bacillus subtilis microparticle capsule, 12˜16% of polyolefin, 80˜87% of calcium carbonate, 2% of dispersant, and 1% of zinc stearate (by weight percentage of dry solid form).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1: The structure diagram of biodegradable plastic masterbatch;

[0042] FIG. 2: The 3D tunnel photograph of Bacillus subtilis SK01 in a plastic product.

[0043] FIG. 3: The 3D tunnel photograph of expanded foam of plastic caused by the interaction of microbial enzymes produced by Bacillus subtilis SK01 with the plastic;

[0044] FIG. 4: The size of Bacillus subtilis SK01 in a plastic product (3D tunnel photograph);

[0045] FIG. 5: The carbon dioxide release curve during the cellulose degradation of 2012E0568 specimen and reference material.

[0046] FIG. 6: The biological decomposition curve during the cellulose degradation of 2012E0568 specimen and reference material.

DETAILED DESCRIPTION

Embodiments

Embodiment 1: Acquisition of Bacillus subtilis SK01 and Relevant Characteristic Detection

[0047] The native strain of Bacillus subtilis SK01 adopted in the disclosure was taken from dry rice straw, and the procedure for screening is as follows: [0048] 1. Optimization of media and screening of conditions for culture of Bacillus Subtilis SK01 [0049] (1) C source basic medium: yeast extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, pH 7.2-7.4. [0050] (2) N source basic medium: glucose 5 g/L, NaCl 5 g/L, pH 7.2-7.4.

[0051] Culture the selected strain in C source basic medium, with glucose as the C source, prepare media with different glucose concentrations, and for each medium, dispense 25 mL in a 250 mL conical flask. Investigate the effects of glucose concentration on spore formation of the native strain.

[0052] With glucose concentration as the C source, to N source basic medium, add organic nitrogen: peptone, yeast extract, corn pulp powder, soybean powder, soybean cake powder, peanut cake powder, fish meal and yeast powder, 10.0 g/L each; inorganic nitrogen: urea, NH4Cl and (NH4)2 SO4, 20.0 g/L each. For each medium, dispense 25 mL in a 250 mL conical flask, and investigate the effects of different nitrogen sources on spore formation of the native strain.

[0053] To the optimized carbon source and nitrogen source media, add NaCl, CaCO3, MgSO4.Math.7H2O, KH2PO4, K2HPO4, and K2HPO4+KH2PO4 respectively to prepare media. For each medium, dispense 25 mL in a 250 mL conical flask, and investigate the effects of different inorganic salts on spore formation of the native strain.

[0054] In the optimized media, the effects of inoculum size and aeration on the number of fermentation bacteria and the spore formation were investigated by a single factor test. With the pH value of media set at 7.0, for each medium, 25 mL was dispensed into a 250 mL conical flask, and inoculated with the strain with a spore rate above 90% screened in the preceding steps, so that the spores in the shake flasks achieved 104, 105, 106, and 107 orders of magnitude respectively. After 24 h of culture in the shake flask, the effect of inoculum size on the spore formation of bacillus was investigated. For each medium, 25 mL and 50 mL were separately dispensed into 250 mL shake flasks to investigate the effects of aeration on bacterial growth and spore formation.

[0055] After several rounds of screening, a strain of Bacillus subtilis SK01 that could produce acidic enzymes under the activated condition of organic matter was obtained. The strain may produce a variety of proteases (especially alkaline protease), saccharase, lipase and amylase depending on the environmental nutrient sources.

[0056] A study found that Bacillus subtilis SK01 was implanted in a polymer plastic, and affected by pH value or temperature change in the environment or ultraviolet radiation, the plastic was physically deteriorated, resulting in the release of Bacillus subtilis SK01 implanted in the polymer plastic due to the deterioration of carrier (For example: After the food package is broken, the food may go moldy for mold parasitism). The released Bacillus subtilis SK01 rapidly expands and grows with organic matter in the environment as the nutrient source. There are different kinds of organic matter in the environment, and therefore, the biological enzymes formed after conversion are diverse. For the polymer originally used as the carrier of Bacillus subtilis SK01, the organic matter that enters the nutritional food chain synchronously is decomposed into carbon dioxide, inorganic salts, and water at last. As protected by excipients, Bacillus subtilis SK01 may own high temperature resistance as well as properties that contribute to plastic foaming.

[0057] The name under Classification and Nomenclature for Collection is Bacillus Subtilis SK01.

Embodiment 2: Preparation and Degradation Detection of Biodegradable Plastics

[0058] 1. Preparation of Bacillus subtilis Microparticle Capsule

[0059] The mixed Bacillus subtilis SK01 microorganism (49˜53% by weight percentage of solid matter) and chitosan (45˜47% by weight percentage of solid matter) underwent the following process: 43-45° C. low temperature drying.fwdarw.grinding.fwdarw.1250˜1500 mesh screening.fwdarw.fine grinding (to sub-micron level, 10-8 m).fwdarw.adding methyl silicone oil (1˜4% by weight percentage of solid matter) for compounding.fwdarw.completion of coating.fwdarw.Bacillus subtilis microparticles capsule.

[0060] Chitosan may be obtained from shrimp and crab chitosans after acid dissolution or purchased directly. It is characterized by high temperature resistance and polymer formation at a high temperature. After mixed with Bacillus subtilis SK01, it can effectively protect the activity of Bacillus subtilis SK01 from being lost at a temperature below 340° C.

[0061] Adding methyl silicone oil for compounding is to prevent the mixture of Bacillus subtilis SK01 and chitosan from volatilization when the powder reaches the sub-micron level (10-8) after low temperature drying.fwdarw.grinding.fwdarw.sifting.fwdarw.fine grinding. The temperature for decomposition of methyl silicone oil is about 316° C., and with the increasing substitution of methyl group by propyl group, the decomposition temperature is also increasing. When the content of propyl group is 30%, the decomposition temperature is up to 400° C.

[0062] Because the plastic products need a high temperature above 170° C. for melting and polymerizing in the preparation process, with chitosan polymer as a parasitic carrier, methyl silicone oil is used for coating, thus achieving high dispersion, high cohesion and high temperature resistance to protect their activity from being lost.

[0063] Bacillus subtilis microparticle capsule can withstand 340° C./0.35 hr of high temperature without destruction of composition under dry and hot conditions.

[0064] 2. Preparation of Biodegradable Plastic Masterbatch

[0065] Place 0.3˜1% of Bacillus subtilis microparticle capsule, 12˜15% of polyolefin resin plastic, 75˜81% of calcium carbonate powder with fineness above 1250 mesh, 2% of dispersant and 1% of zinc stearate in a bucket, stir and mix well.

[0066] Send the mixture to an internal mixer at high temperature for 25˜35 minutes of banburying under 170˜202° C., and then perform 2˜5 min of granulation by a screw extruder at 190˜202° C. to obtain grains.

[0067] The biodegradable plastic masterbatch may be obtained after air cooling, sifting, aggregating, weighing and bagging of the grains.

[0068] As shown in FIG. 1, the microstructure of biodegradable plastic masterbatch is in a form of balloon encapsulation. In the preparation process, Bacillus subtilis SK01 absorbs gas itself, and the gas is blocked by polymer due to banburying and melting, thus forming a balloon foam. As the temperature increases, the balloon is further inflated while Bacillus subtilis SK01 in the balloon floats in the gas. When Bacillus subtilis SK01 approaches the high temperature of balloon wall, it moves to the space at a lower temperature instinctively. Please refer to FIGS. 2-4, where the 3D tunnel photographs show that the balloon volume is 200 to 250 nm, while the volume of Bacillus subtilis SK01 is 8 to 25 nm. Therefore, the gas in the balloon can maintain Bacillus subtilis SK01 at a constant body temperature at high temperatures indirectly or directly, and through physical regulation, reduce the effect of temperature on Bacillus subtilis SK01, or even its loss.

[0069] The biodegradable plastic masterbatch may reduce the difference with polyolefin plastics in specific gravity. During the banburying and melting process of Bacillus subtilis SK01 and polyolefin resin plastic, the oxygenophilic expansion and growth at 65° C.˜75° C. make polyene plastics produce microbubbles, resulting in expanding and foaming of polyene plastics. Under the same volume, the specific gravity of polyene plastics added with Bacillus subtilis SK01 is 0.919˜0.926, which is 0.04 lower than polyene plastics without Bacillus subtilis SK01 (specific gravity: 0.923˜0.93).

[0070] Relevant detection found that the plastics made of biodegradable plastic masterbatch achieved 49.11% of total degradation of organic solids after 153 days in an environment for composting or soil burial, and were slowly degraded by sunlight (photodegradation) or immersion in water. Under an environment other than that mentioned above, the plastics made of biodegradable plastic masterbatch are consistent with the non-degradable plastics in shelf life, which is more than 1 year.

[0071] 3. Degradation Detection

[0072] The biodegradable plastic masterbatch may be used to manufacture polyene products, e.g., films, bags, blister products, hard plastics, or adjust the dosage of polyolefin materials between 50˜95% and of biodegradable plastic masterbatch between 5˜50% depending on the physical demands. The values and value ranges in percentage in this embodiment are all by weight percentage.

[0073] According to three experiments, each two-year verification (certified by Plastic Products Quality Supervision and Inspection Center (Beijing), GSJ [2013] C0292) showed that polyolefin materials add with more than 5% of biodegradable plastic masterbatch may induce the performance of biodegradable polyolefin organics, and the proportion for addition is inversely proportional to the physical demand. The more the biodegradable plastic masterbatch, the stronger the induced biodegradability, and the poorer the transverse/longitudinal tension and extension force as physical properties. According to a physical test, with 0.008 mm of plastic film thickness and 5% of masterbatch proportion as the base points, for every 0.001 mm increment, the addition ratio can be increased by 1˜1.2%, with 50% as the upper limit.

[0074] For a 0.035 mm diaphragm specimen made of 54% of finished polyolefin material and 46% of biodegradable plastic masterbatch, the 153-day polyolefin degradation rate of polyolefin material (with calcium carbonate as the inorganic matter) reached 90.9%. The reference material was cellulose.

TABLE-US-00002 TABLE 1 Basic characteristics of the specimen Total Theoretical carbon Dry organic dioxide release Appear- solid Water carbon in (CO.sub.2g/100 g dry Sample ance Content Content dry solids sample) Specimen diaphragm 98.48% 1.52% 58.90% 215.97 Reference Powder 99.06% 0.94% 43.03% 157.78 material

TABLE-US-00003 TABLE 2 Carbon dioxide release and bio-decomposition rate CO.sub.2 Bio- CO.sub.2 release of decomposition Bio- release reference rate CO.sub.2 release decomposition of blank material, of reference of specimen, rate of Days test, g g material, % g specimen, % 153 56.0 149.3 94.6 106.1 49.1

TABLE-US-00004 TABLE 3 Carbon dioxide output and bio-decomposition rate CO.sub.2 output, g Decomposition rate, % 2012E0568 Cellulose 2012E0568 2012E0568 Days Blank Cellulose Specimen (actual) Specimen Cellulose Specimen 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 15.04 34.53 26.15 19.49 11.11 12.35 5.14 20 41.29 72.17 54.31 30.88 13.02 19.57 6.03 30 51.18 109.01 81.86 57.83 30.68 36.65 14.21 40 52.84 150.88 113.18 98.04 60.34 62.14 27.94 50 53.14 171.81 128.83 118.67 75.69 75.21 35.05 60 53.44 179.70 135.77 126.26 82.33 80.02 38.12 70 53.73 187.59 145.94 133.86 92.21 84.84 42.70 80 54.03 195.49 151.64 141.46 97.61 89.66 45.20 90 54.33 201.34 153.97 147.01 99.64 93.17 46.14 100 54.62 202.87 154.91 148.25 100.29 93.96 46.44 110 54.92 203.45 156.55 148.53 101.63 94.14 47.06 120 55.21 203.90 159.89 148.69 104.68 94.24 48.47 130 55.46 204.35 161.14 148.89 105.68 94.37 48.93 140 55.68 204.80 161.59 149.12 105.91 94.51 49.04 150 55.95 205.25 161.97 149.30 106.02 94.63 49.09 153 56.05 205.38 162.12 149.33 106.07 94.64 49.11

[0075] FIG. 5 shows the carbon dioxide release curves of 2012E0568 specimen and cellulose as the reference material during degradation.

[0076] The biodegradable plastic masterbatch of this disclosure induces biodegradable polyolefin polymer based on the polyolefin material implanted with highly processed Bacillus subtilis SK01 as the main principle of action. With high temperature resistance, the components of Bacillus subtilis SK01 will not be destroyed at high temperatures for granulation, extrusion, injection, film formation or bagging. After use, the bags and films are buried in the soil or compost, and microbial enzymes produced through the growth and proliferation of Bacillus subtilis SK01 induce indigenous bacteria to gather and decompose, so that the bags and films are cracked into fine sheets (4˜5 cm), and through ecological chemical chelation, decomposed into powder particles (0.01˜0.1 mm), which are swallowed and digested by the ecological metazoan, and at last, converted into carbon dioxide, water and calcium carbonate or reduced to harmless substances in the ecological environment.

[0077] The products added with biodegradable plastic masterbatch passed tests of FDA and RoHS, tests for 12 heavy metals, 21 chemical toxicity tests, yellowing level 4 test, and the highest-level test of horizontal and longitudinal tension, and thus, a relevant high-quality evaluation on degradation performance, safety and physical properties was obtained.

[0078] For the products added with biodegradable plastic masterbatch, the test results of lead, cadmium, mercury, hexavalent chromium, polybrominated biphenyl (PBB), polybrominated diphenyl ether (PBDE) were within the limits specified in Appendix II of Directive 2011/65/EU as revision of RoHS Directive 2002/96/EC (EU).

[0079] According to the regulatory requirements of the US FDA, the maximum extractable amount of polyethylene in biodegradable plastic masterbatch as a component used for coating in contact with food was determined. For the test method, refer to US FDA 21 CFR 177.1520 d(3)(ii)&d(4) (ii). The test results are as follows:

TABLE-US-00005 Conventional Maximum Detection simulation allowable value of solution Time Temperature limit sample N-hexane 2 hr 50° C. 53% (w/w) 3.7 (w/w) xylene 2 hr 25° C. 75% (w/w) 5.7 (w/w)

[0080] The packing bag added with biodegradable plastic masterbatch is 700±5 mm long and 510±5 mm wide with bilateral stitches. The number of monofilaments within 100 mm is no less than 47. The mechanical performance test results and physical properties are as shown in the following table:

TABLE-US-00006 Tensile load (N/50 mm) not less than Mass per In the In the In the In the In the unit area radial zonal direction of direction of direction of Peel force (g/m.sup.2) direction direction adhesion hemming hemming bottom (N, 30 mm) ≥75 450 450 300 250 200 ≥3.0

[0081] Firmness: With 5 packing bags randomly selected, the failure rate after 2.5 m±2 mm high altitude free fall: ≤4.

[0082] The temperature applicable for the packing bags: ≥100° C.

[0083] The influence of paper bag material on cement strength: 3-day flexural strength ratio ≥93%, 3-day compressive strength ratio ≥95%.

[0084] Moisture-proof performance of the packing bags: 3-day compressive strength ratio ≥90%.

[0085] The tensile test results of monofilament: At least 80% passed the tension test of monofilament.

[0086] The packing bags prepared with biodegradable plastic masterbatch and polyethylene as raw materials, the product inspection results based on GB13735-92 standard are as shown in the following table:

TABLE-US-00007 Inspection Individual Name Item Standard requirements results evaluation Specification Width limit ±15 +8 Superior deviation, mm quality Thickness limit ±0.002 +0.002 Superior deviation, % quality Average thickness ±10 +5 Superior deviation % quality Average thickness, 0.008 0.0084 Superior mm quality Appearance Watermark and Does not affect use There is Superior cloud no quality Bubble perforation There is no There is Superior and rupture no quality Impurities, fish See Standards for details There is Superior eyes and stiff no quality blocks Streaks and tears Flat tears should not form There is Superior straight lines no quality Roll paper core for 20 ± 5 mm 24 Superior diaphragm quality exposed Folding and There is Superior misalignment no quality Bulges Not allowed There is Superior no quality Physical Mass Tensile load, N Longitudinal/transverse ≥ 1.6 2.2/2.0 Superior property per unit quality     Elongation at Longitudinal/transverse ≥ 160 448/672 Superior break, % quality Right angle load, Longitudinal/transverse ≥ 0.6 1.2/1.5 Superior N quality Tensile load, N Longitudinal/transverse≥ // // Elongation at Longitudinal/transverse≥ // // break, % Right angle load, Longitudinal/transverse≥ // // N Functional Light // // // performance transmittance, % Haze, % // // // Conclusion Superior quality indicates data missing or illegible when filed

[0087] Different from grain based materials (The main biodegradable plastics based on natural substances like starch include the following products: polylactic acid (PLA), polyhydroxyalkanoate (PHA), starch plastics, bioengineering plastics, biological general-purpose plastics like polyolefin and polyvinyl chloride, and etc.), the biodegradable plastic masterbatch, with the mechanism of inducing biodegradable polyene polymer, may be used to produce biodegradable plastic products without any change, increase or modification of production process or equipment. The products own a strong market competitiveness due to the affordable cost.

[0088] This document has utilized specific cases to elaborate the inventive concept, while the above embodiments are only to help understand the core idea of the disclosure. It should be noted that for ordinary technical personnel in the field, any significant modification, equivalent replacement, or other improvements without deviation from the conception of the disclosure shall be included in the scope of protection for the disclosure.