CN114672430A

CN114672430A - Special compound microbial inoculum for biodegradable plastic compost treatment

Info

Publication number: CN114672430A
Application number: CN202210231979.8A
Authority: CN
Inventors: 王微山; 李林林; 孔祥坤; 杨学军; 齐永润; 郭风; 邓宁; 徐正; 杨晓煜; 孙筱辰
Original assignee: Shandong Institute for Product Quality Inspection
Current assignee: Shandong Institute for Product Quality Inspection
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-06-28
Anticipated expiration: 2042-03-09
Also published as: CN114672430B

Abstract

The invention relates to a high-efficiency compost composite microbial inoculum, and relates to the technical field of biological plastic degradation. The high-efficiency compost composite microbial inoculum comprises Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Brevibacillus paraburgensis), Bacillus cereus (Bacillus cereus), Rhodopseudomonas palustris (Rhodopseudomonas palustris) and lysine sphaericus (Lysinibacillus sphaericus). The invention screens out the high-efficiency compound microbial inoculum for composting suitable for biodegradable plastics, optimizes the proportion of the compound microbial inoculum through a biological decomposition rate test, greatly shortens the degradation time and provides data support for the standardized popularization among laboratories.

Description

Special compound microbial inoculum for biodegradable plastic compost treatment

Technical Field

The invention relates to the technical field of biodegradable plastic degradation, in particular to a special compound microbial inoculum for biodegradable plastic composting treatment.

Background

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Under the influence of global 'plastic limitation and plastic prohibition', degradable plastics are regarded as one of important ways for realizing sustainable development of the environment by various countries in the world, and the degradable plastics in the market are very many in variety and wide in quantity at present, and relate to the fields of express logistics packaging, disposable tableware, shopping bags, mulching films and the like. At present, all kinds can beThe degradable material becomes the focus of enterprise pursuit, especially the biodegradable plastic, which means that under the condition of nature such as soil and/or sandy soil, and/or under the specific condition such as composting condition or anaerobic digestion condition or in the aqueous culture solution, the degradation is caused by the action of the microorganisms existing in nature, and finally the complete degradation is changed into CO₂Or/and mineralized inorganic salts of methane, water and its elements contained, and new plastics of biomass. The biodegradable material industry projects are all over the country, and the varieties which are industrially produced include more than ten kinds of thermoplastic starch, polylactic acid (PLA), polybutylene adipate-butylene terephthalate copolymer (PBAT), polybutylene succinate (PBS), Polyhydroxyalkanoate (PHA), Polycaprolactone (PCL) and the like.

Biodegradable plastic products meet the requirement of degradation besides the service performance, the biodegradation is a well-known degradation way of thorough decomposition at present, and any degradable plastic enters a biodegradation stage, so that a standard system for testing the degradation performance internationally and domestically surrounds a biodegradation test, the biodegradation is divided into two modes of aerobic biodegradation and anaerobic biodegradation according to whether the degradation environment is aerobic or not, aerobic biodegradation products are water, carbon dioxide and inorganic mineral substances, products obtained in the anaerobic environment are water, carbon dioxide, methane and inorganic mineral substances, and the method for testing the aerobic biodegradation rate is more easily realized by considering the calculation mode of simulating the degradation environment and collecting carbon. At present, GB/T19277.1-2011 part 1 of a method for measuring the final aerobic biological decomposition capacity of a material under a controlled composting condition is adopted in China: the general method is used as an arbitration method for testing the biological decomposition rate (relative biological decomposition rate), the method is used for testing specific compost materials, temperature, humidity, pH value and other test conditions according to standard regulations, collecting the accumulated release amount of carbon dioxide, verifying the effectiveness of the test through reference and blank experiments, and according to the material quality of products, the test period is about 45 days to 180 days, the test period is long, and the test environment requirements are complicated and harsh. Although blank, reference and parallel tests are considered in the test process to ensure the reproducibility and the parallelism of the test, specific microbial inoculum used in compost materials is not determined in the standard, the microbial inoculum plays a key role in the compost degradation process, different microbial inocula are used in different laboratories, so that the same degradation materials are detected according to the standard to obtain different degradation periods, and the data deviation among the laboratories is too large.

During the composting process, microorganisms can produce specific hydrolytic enzymes to degrade organic matters in raw materials, and cellulase, urease, protease, amylase and the like are main hydrolytic enzymes. The degradation rate of organic matters is in positive correlation with the activity of corresponding hydrolase, and the enzyme production activity of microorganisms in the compost directly influences the quality and the period of the compost.

It is also worth noting that the composting biological process, the type of microorganism that plays a major role in the degradation of bioplastics at different stages of the composting process, requires control of factors that affect the microorganism, which determine the activity level of the microorganism and the enzyme production activity, and thus affect the degradation rate and quality. Meanwhile, the degradation of plastics is caused by physicochemical changes caused by polymer chain breakage, chemical conversion and the like under the influence of environmental factors such as light, heat, humidity, chemical conditions, biological activity and the like, and finally the performance and functions of polymers are deteriorated, so the reaction process is complex, the influence factors are numerous, different enzymes are influenced by the reaction environment, the enzymes play a role through different mechanisms, the types of strains playing a key role in the degradation process and the enzyme activity have unpredictability, the strains which do not have high enzyme activity per se can keep the performance per se in the biodegradation process, and the strains which cannot play a key degradation process can not improve the degradation efficiency of the biological plastics even if having high enzyme activity no matter the influence factors are numerous in the composting fermentation process. Only if the two have the characteristics that the bacterial strains playing an important role in biodegradation in the composting process can also keep high activity, the excellent degradation effect of the biological plastics can be achieved.

Based on the above, the development of a high-efficiency special compound microbial inoculum for the biodegradable material compost is urgent.

Disclosure of Invention

In order to overcome the problems, the invention designs a compound microbial inoculum for improving the degradation rate of the biological plastics, establishes a high-flux biological degradation enzyme biopsy platform for the biological degradation plastics, and evaluates the functional information of each strain from the aspect of degradation enzyme. Corresponding high-yield enzyme strains are selected according to biodegradable plastic components in the composting raw materials, a composting composite microbial inoculum is constructed, the application effect of the microbial inoculum is verified through a composting test, whether a method for constructing the microbial composite microbial inoculum is feasible or not is verified, and the method has important significance for the development of a composite microbial inoculum construction method.

In a first aspect of the present disclosure, a high efficiency composting composite microbial inoculum is provided, which comprises Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Brevibacillus paraburgensis), Bacillus cereus (Bacillus cereus), Rhodopseudomonas palustris (Rhodopseudomonas palustris), and Bacillus sphaericus (Lysinibacillus sphaericus).

In a second aspect of the present disclosure, a preparation method of the complex microbial inoculant is provided, which includes: preparing a culture medium, inoculating strains with corresponding content ranges into the culture medium, standing at 20-37 ℃, and performing expanded culture of the strains; further, the culture medium is a brown sugar culture medium which comprises 3.6g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium chloride, 0.2g of magnesium sulfate heptahydrate, 4 mu M of ferric chloride and 20g of brown sugar, and is sterilized at 121 ℃ for 20 min; the inoculation amount is 3% -10%, further, the inoculation amount is 8%, and the standing temperature is 25 ℃.

In a third aspect of the disclosure, a method for using the compost composite microbial inoculum is provided, which comprises the following steps: adding a biodegradable plastic product in a composting environment, adding a compound microbial inoculum after expanded culture to form a mixed material to be treated, and carrying out a composting test; further, the composting test conditions are: the temperature is 58 ℃, the humidity is 100%, and the pH range is 6-8.

In the fourth aspect of the disclosure, the application of the compost composite microbial inoculum in degrading the biological plastics is improved, and further, the biological plastics are the biological plastics consisting of PLA + PBAT + starch or PLA + PBAT + CaCO₃The formed biological plastic.

The fifth aspect of the disclosure provides an application of the compost composite microbial inoculum in degrading domestic organic garbage, and further, the domestic organic garbage is kitchen garbage.

In a sixth aspect of the disclosure, an application of the compost composite microbial inoculum on daily chemical products is provided, wherein the daily chemical products comprise wet tissue liquid, nursing liquid and the like.

One or more specific embodiments of the present disclosure achieve at least the following technical effects:

the present disclosure provides a high efficiency compost complex microbial inoculum, which can have strong synergistic effect, and can efficiently degrade the biological plastics in a short time. The reason why the above-described effects are produced is that: the synergistic effect of the strains can increase the activity of substrate microorganisms, realize function complementation and synergistic metabolism, shorten the early-stage time of fermentation, enter a high-temperature stage in a short time and realize quick decomposition, thereby improving the degradation efficiency and the composting quality.

The method screens out the efficient compound microbial inoculum for composting suitable for biodegradable plastics, optimizes the proportion of the compound microbial inoculum through a biological decomposition rate test, greatly shortens the degradation time, and provides data support for the standardized popularization among laboratories.

The utility model provides a high-efficient compost composite bacterial agent can effectively degrade domestic waste, especially rubbish from cooking, and the treatment process of rubbish from cooking among the prior art adopts landfill and burning mostly, causes the pollution problem of environment and soil, and the composite bacterial agent of this application can realize degrading rubbish from cooking fast, and the degradation rate is up to 97.6%, and the treatment process is simple, the practicality is strong.

The utility model provides a high-efficient compost composite bacterial agent, has excellent antibacterial performance, and wet tissue liquid among the prior art etc. mostly adds the antiseptic and carry out the bacteriostasis, and not only antibacterial action is limited and have certain irritability, and this application has realized nontoxic high antibacterial performance, can reach 96.8% and 97.2% respectively to the antibacterial performance of staphylococcus aureus and candida albicans.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a graph of a) CO2 release curve and b) biological decomposition rate curve of the bioplastic PLA + PBAT + starch in the commercial microbial inoculum condition in example 1 of the present invention;

FIG. 2 is a graph of a) a CO2 release curve and b) a biological decomposition rate curve of the bioplastic PLA + PBAT + CaCO3 under a commercial microbial inoculum condition in example 1 of the invention;

FIG. 3 is a graph showing a) a CO2 release curve and b) a biological decomposition rate curve of the bioplastic PLA + PBAT + starch under the condition of the high-efficiency BP bacterial agent in example 1 of the present invention;

FIG. 4 is a graph of a) CO2 release curve and b) biological decomposition rate curve of the bioplastic PLA + PBAT + CaCO3 under the condition of high-efficiency BP bacteria agent in example 1 of the invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, there are few reports in the prior art that the strain can rapidly enter a fermentation temperature stage, shorten the early-stage fermentation time, and screen and obtain strains which play a key role in the biodegradation process and keep high activity. Therefore, the present disclosure provides a compound microbial inoculum for increasing the degradation rate of the biological plastics, which can effectively solve the technical problems in the prior art.

In a first aspect of the present disclosure, a high efficiency composting composite microbial inoculum is provided, which comprises Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Brevibacillus paraburgensis), Bacillus cereus (Bacillus cereus), Rhodopseudomonas palustris (Rhodopseudomonas palustris), and Bacillus sphaericus (Lysinibacillus sphaericus);

in a typical embodiment, the high efficiency composting complex microbial inoculum further comprises Enterobacter aryabhattai (Enterobacter asbuiae) and Bacillus licheniformis (Bacillus licheniformis); further, Corynebacterium glutamicum (Corynebacterium glutamicum) is also included.

The strains used in the present disclosure are production strains allowed to be used in the appendix of the general technical guidelines on the biological safety of microbial feeds of the Ministry of agriculture, preferably, the strains are CICC 10081 for Bacillus amyloliquefaciens, CICC 10343 for Bacillus brevis, CICC 10041 for Bacillus cereus, CICC 23812 for Rhodopseudomonas palustris, CICC 23697 for Lysinibacillus sphaericus, CICC 10013 for Enterobacter aryabhattai, CICC 10092 for Bacillus licheniformis and CICC 10058 for Corynebacterium glutamicum, and are all purchased from the China center for the preservation and management of industrial microbial strains.

In the cognition in the field, the enterobacter aryabhattai with the strain number CICC 10013 has the effect of producing 2, 3-butanediol, and the corynebacterium glutamicum with the strain number CICC 10058 is a glutamic acid producing bacterium, but the inventor unexpectedly finds that the degradation efficiency can be obviously improved in a subsequent biological plastic degradation experiment after the corynebacterium glutamicum is compounded with other strains, and deserves that the degradation efficiency can be improved by 30% by adding the enterobacter aryabhattai, the bacillus licheniformis and the corynebacterium glutamicum, so that the three microbial agents and the five strains can show strong synergistic effect in a composting fermentation process, and the biological plastic can be efficiently degraded in a short fermentation time. The complex microbial inoculum can play a key degradation role in the process of biodegradation of plastics, and can keep high-efficiency enzyme activity, so that the relation between the bacterial strain and decomposition of carbohydrates in the composting process and pathogenic bacteria growth inhibition can be further explained. Meanwhile, in the three strains of the enterobacter aryabhattai, the bacillus licheniformis and the corynebacterium glutamicum, the corynebacterium glutamicum obtained through the degradation result verification can play a more critical role, so that the enzymes generated by the corynebacterium glutamicum in the degradation and fermentation of the biological plastics can more effectively cooperate with other enzymes, the compost maturity is promoted, and the compost period is shortened.

In a typical embodiment, the strain comprises the following components by mass: bacillus amyloliquefaciens (Bacillus amyloliquefaciens): 22% -35%, bacillus brevis (Brevibacillus parabrevibensis): 7% -15%, Bacillus cereus (Bacillus cereus): 8% -12%, Rhodopseudomonas palustris (Rhodopseudomonas palustris): 10% -15%, lysine bacillus sphaericus (Lysinibacillus sphaericus): 10% -15%; further, Enterobacter aryabhattai (Enterobacter alburiae) is included: 7% -14%, Bacillus licheniformis (Bacillus licheniformis): 7% -14%, Corynebacterium glutamicum (Corynebacterium glutamicum): 7% -14%; furthermore, the strain comprises the following mass components: bacillus amyloliquefaciens (Bacillus amyloliquefaciens): 25%, bacillus brevis (Brevibacillus parabrevibensis): 10%, Bacillus cereus (Bacillus cereus): 10%, Rhodopseudomonas palustris (Rhodopseudomonas palustris): 12.5%, lysine bacillus sphaericus (Lysinibacillus sphaericus): 12.5%, Enterobacter aryabhattai (Enterobacter albureae): 10%, Bacillus licheniformis (Bacillus licheniformis): 10%, Corynebacterium glutamicum (Corynebacterium glutamicum): 10 percent.

In the process, the selection of the content influences the type, the content and the activity of the enzyme after the fermentation of the strains, and the action modes of the strains with different proportions in the degradation process of the biological plastics are certainly different, which directly influences the final degradation effect. The inventor finds out through continuous screening and optimization that the strains with the content combination can achieve a good action mode, namely, the strains can be quickly decomposed.

In a typical embodiment, the method for screening the complex microbial inoculum comprises the following steps:

(1) preparing compost raw materials;

(2) screening high-temperature degradation bacteria: the method comprises the steps of primary screening and secondary screening, wherein the primary screening comprises the steps of carrying out high-temperature enrichment culture on a sample in the step (1), separating by using a gradient dilution method to obtain a high-temperature-resistant pure strain, then inoculating the high-temperature-resistant pure strain into a culture medium, judging the capability of the strain to degrade a substrate, and selecting a strain with high degradation capability, and the secondary screening comprises the steps of culturing the strain obtained by the primary screening and measuring the enzyme activity;

(3) and (3) carrying out strain identification on the strains obtained by the secondary screening in the step (2).

In a typical embodiment, in step (1), the compost raw material comprises vermiculite and culture solution, the mass of the vermiculite: the volume ratio is 1: 3, the culture solution consists of mineral solution, nutrient components, urea, corn starch, cellulose and compost extract, and the proportion is as follows: 500 mL: 13 g: 5.8 g: 20 g: 20 g: 500 mL; further, the mineral solution consists of KH2PO4, MgSO4, CaCl2, NaCl and a trace element solution, and the proportion of the solution is as follows: 1 g: 0.5 g: 1mL of: 1mL of: 1mL, and the solubility of CaCl2 is 10%; furthermore, the trace element solution consists of H3BO3, KI, FeCl3, MnSO4, (NH4)6MO7O24 and FeSO4, and the proportion is as follows: 500 mg: 100 mg: 200 mg: 400 mg: 200 mg: 400 mg.

In a typical embodiment, the compost extracting solution is a high-efficiency complex microbial inoculum, a brown sugar culture medium and distilled water, and the weight ratio of the compost extracting solution to the distilled water is 0.2: 1: 100 by mass ratio.

In a typical embodiment, in the step (2), enrichment culture is carried out at a high temperature of 60 ℃ during primary screening, the enrichment culture is inoculated into a hydrolysis culture medium, and whether a transparent hydrolysis ring appears around a colony is observed; if a hydrolysis ring appears, the substrate is hydrolyzed, the strain has the capacity of degrading the substrate, and the capacity of degrading organic matters is shown as the formula Up ═ D/D)²Calculating, wherein the magnitude of the Up (organic matter degradation capacity) value reflects the strength of the hydrolysis capacity of the strain, D is the diameter (mm) of a transparent ring, D is the diameter (mm) of a bacterial colony, and strains with the Up value larger than 25 (cellulose), 12 (protein) and 15 (starch) are selected for further research. The applicant has found through a large number of experiments that the above strains are selected: strains with different up values directly influence the enzyme production activity and enzyme production types of the strains in the composting fermentation process and play a key role in the degradation of biological plasticsThe kind of the same. It was found in the constant screening that strains with the above up-value could be screened for the strain class that plays a key role in the degradation of bioplastics during composting and could have high enzyme activity.

In a typical embodiment, the hydrolysis medium in step (2) comprises a cellulose congo red medium, a protein hydrolysis medium and a starch hydrolysis medium, wherein the cellulose congo red medium is: CMC-Na 2.0g, (NH)₄)₂SO₄2.0g，MgSO₄·7H₂O0.5g，K₂HPO₄1.0g, NaCl0.5g, Congo red 0.4g, agar 20g, deionized water 1000mL, pH 7.0, 121 ℃ sterilization for 30min, the proteolysis culture medium is: dissolving 42g hydrolyzed casein agar culture medium in 1000mL distilled water under heating, sterilizing at 121 deg.C for 15min, wherein the starch hydrolyzed culture medium comprises 10g soluble starch, 10g peptone, 5g yeast powder, 10g sodium chloride, 15g plant gel, 0.05-0.1 g Trimeryl blue and 1000mLH₂O, pH 7.0, sterilizing at 121 deg.C for 30 min.

In a typical embodiment, in the step (2), in the secondary screening process, the strains obtained by primary screening are inoculated into a cellulose, starch and protein liquid fermentation medium, static culture is carried out for 72h at 50 ℃, and then the fermentation liquor is taken and centrifuged for 10min at the rotating speed of 5000r/min to obtain crude enzyme liquid; respectively measuring the cellulase activity, amylase activity and protease activity of the strain by using a DNS method, a modified Yoo method and a welin reagent method; and (3) analyzing the main components of the measurement result by using SAS software, and comprehensively evaluating the degradation capability of the strain.

Further, the cellulose fermentation medium is a bran medium, and comprises: crushing and sieving 90.0g of bran with a 100-mesh sieve, 0.5g of NaCl, 0.3g of K2HPO40, 0.1g of FeSO4 & 7H2O0.1g, 0.1g of MgSO4 & 7H2O0.1 and 1000mL of water, and sterilizing at 121 ℃ for 20 min; the starch fermentation medium comprises: sterilizing 30g of potato glucose agar culture medium and H2O100mL at pH 7.0 at 121 deg.C for 30 min; the protein liquid fermentation medium comprises: 10g of peptone, 15g of plant gel, 0.05-0.1 g of Triflozin blue and 1000mL of H2O 1000, wherein the pH is 7.0, and the peptone is sterilized at 121 ℃ for 30 min.

In an exemplary embodiment, in step (3), the strain identification includes morphological characteristics and part of physiological and biochemical characteristics, strain growth characteristics and 16Sr DNA identification, and the morphological characteristics and part of physiological and biochemical characteristics are: after the strain is purified, observing the bacterial colony characteristics of the strain on a beef extract peptone culture medium, carrying out crystal violet staining on the cultured thallus for 24 hours to judge the morphological characteristics of the strain, and researching partial physiological and biochemical characteristics of the strain; or, the strain growth characteristics comprise: inoculating the strain seed liquid obtained by screening into a beef extract peptone culture medium, changing a certain condition, keeping other conditions unchanged, and researching the influence of temperature, pH, NaCl mass concentration and liquid loading amount on the growth of the strain by measuring the OD600 value of the bacterial liquid; or, the 16Sr DNA identification comprises: using the screened high-efficiency high-temperature degradation bacterium genome DNA as a template, and carrying out PCR amplification by using general primers 27f and 1492r of general bacteria; the method comprises the following steps: ddH2O 37.5.5 μ L; 27f0.5 μ L; 1492r0.5 μ L; 10 × Tap Buffer 5 μ L; dNTP1 μ L; 5 mu L of template; 0.5 mu L of Tap enzyme; the procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 10 s; the annealing temperature adopts gradient temperature, the temperature is respectively 52.1 ℃, 53.5 ℃, 54.7 ℃, 55.6 ℃, 56.9 ℃ and 58.1 ℃, and the annealing is carried out for 40 s; extending for 1.5min at 72 ℃; for 30 cycles, and after the cycle is finished, the extension is carried out for 10min at 72 ℃. Sequencing the PCR product: and performing Blast analysis and comparison on the obtained sequence and a sequence in a GeneBank database to call out a 16Sr DNA gene sequence of a related strain with the highest similarity. The addresses of sequence alignment are: http:// blast.ncbi.nlm.nih.gov/blast.cgi.

In a second aspect of the present disclosure, a preparation method of the complex microbial inoculant is provided, which includes: preparing a culture medium, inoculating the strain with the content range into the culture medium, standing at 20-37 ℃, and performing expanded culture of the strain; further, the culture medium is a brown sugar culture medium which comprises 3.6g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium chloride, 0.2g of magnesium sulfate heptahydrate, 4 mu M of ferric chloride and 20g of brown sugar, and is sterilized at 121 ℃ for 20 min; the inoculation amount is 3% -10%, further, the inoculation amount is 8%, and the standing temperature is 25 ℃.

In a third aspect of the present disclosure, a method for using the compost composite bacterial agent is provided, which includes: adding a biodegradable plastic product into a composting environment, adding a composite microbial inoculum subjected to expanded culture to form a mixed material to be treated, and performing a composting test; further, the composting test conditions are: the temperature is 58 ℃, the humidity is 100%, the pH value is 6-8, and the composting environment is an activated vermiculite composting environment.

In a fourth aspect of the disclosure, the application of the compost composite microbial inoculum in degrading the biological plastics is improved, and further, the biological plastics are biological plastics composed of PLA + PBAT + starch or biological plastics composed of PLA + PBAT + CaCO 3.

The sixth aspect of the disclosure provides an application of the compost composite microbial inoculum in daily chemical products, wherein the daily chemical products comprise wet tissue liquid, nursing liquid and the like.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

1. Materials and methods

Equipment: biological decomposition testing system (DA18, Jinandike instrument Co., Ltd.), Total organic carbon Analyzer (German element)

1.1 compost raw Material

The experimental compost raw material is vermiculite, and the source is as follows: north of river; the particle size is 3-6 mm, and the preparation is required according to the GB/T19277.1-2011 standard 8.6 clause. The ratio of vermiculite to broth was 1:3 (mass/volume), and table 1 is the composition of 1L inoculum. The compost extract is prepared by high-efficiency complex microbial inoculum, a brown sugar culture medium and distilled water according to the mass ratio of 0.2: 1: 100.

TABLE 11 composition of the inoculation solutions

TABLE 21 composition of the mineral solution

TABLE 31 composition of the Trace element solutions

Composition (I)

H₃BO₃

KI

FeCl₃

MnSO₄

(NH4)₆MO₇O₂₄

FeSO₄

Content (c) of

500mg

100mg

200mg

400mg

200mg

400mg

1.2 microbial culture Medium

1) The hydrolysis culture medium comprises the following components in percentage by weight: CMC-Na 2.0g, (NH4)2SO42.0g, MgSO4 & 7H2O 0.5.5 g, K2HPO41.0 g, NaCl 0.5g, Congo red 0.4g, agar 20g, deionized water 1000mL, pH 7.0. Sterilizing at 121 deg.C for 30 min. Proteolytic medium: hydrolyzed casein agar medium 42g, dissolved in 1000mL distilled water under heating, and sterilized at 121 deg.C for 15 min. The starch hydrolysis culture medium comprises 10g of soluble starch, 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 15g of plant gel, 0.05-0.1 g of Triflozin blue and 1000mL of H2O 1000, the pH value is 7.0, and the starch hydrolysis culture medium is sterilized at 121 ℃ for 30 min.

2) Fermentation medium

Bran culture medium: pulverizing, sieving with 100 mesh sieve, testa Tritici 90.0g, NaCl 0.5g, K2HPO40.3 g, FeSO 4.7H 2O 0.1.1 g, MgSO 4.7H 2O 0.1.1 g, water 1000mL, and sterilizing at 121 deg.C for 20 min.

Starch culture medium: potato dextrose agar medium 30g, H2O 100mL 100, pH 7.0, sterilized at 121 ℃ for 30 min.

Protein liquid medium: 10g of peptone, 15g of plant gel, 0.05-0.1 g of Triflozin blue and 1000mL of H2O 1000, wherein the pH is 7.0, and the peptone is sterilized at 121 ℃ for 30 min.

A brown sugar culture medium: 3.6g of dipotassium phosphate, 1g of monopotassium phosphate, 1g of ammonium chloride, 0.2g of magnesium sulfate heptahydrate, 4 mu M of ferric chloride and 20g of brown sugar, and sterilizing for 20min at 121 ℃;

Seed culture medium: 30g/L glucose, 6g/L trypsin, 560mg/L K2HPO4, 750mg/L NaH2PO 4.2H2O, 750mg/L Na2HPO 4.

1.3 screening of high-temperature degrading bacteria

1.3.1 preliminary screening

The compost raw materials are subjected to enrichment culture at a high temperature of 60 ℃, and are separated by a gradient dilution method to obtain high-temperature-resistant pure strains. And respectively inoculating the separated strains into a hydrolysis culture medium, and observing whether a transparent hydrolysis ring appears around the bacterial colony. If the hydrolysis ring appears, the substrate is hydrolyzed, the strain has the capability of degrading the substrate, and the degradation capability of the organic matters is shown in the formula of Up ═ D/D)²The calculation is carried out, the magnitude of the Up (organic matter degradation capacity) value reflects the strength of the hydrolysis capacity of the bacteria, wherein D is the diameter (mm) of the transparent ring, and D is the diameter (mm) of the bacterial colony. Strains with up values greater than 25 (cellulose), 12 (protein) and 15 (starch) were selected for further study.

1.3.2 double sifting

Inoculating the strains obtained by primary screening into a cellulose, starch and protein liquid fermentation medium, carrying out static culture at 50 ℃ for 72h, and centrifuging the fermentation liquor for 10min at 5000r/min to obtain a crude enzyme solution. The cellulase activity, amylase activity and protease activity of the strains were determined by the DNS method, the modified Yoo method and the Fulin reagent method, respectively. And (3) analyzing the main components of the measurement result by using SAS software, and comprehensively evaluating the degradation capability of the strain.

1.4 identification of strains

1.4.1 morphological characteristics and partial physiological and biochemical characteristics

After the strain is purified, the bacterial colony characteristics of the strain on a beef extract peptone culture medium are observed, the thalli cultured for 24 hours are subjected to crystal violet staining to judge the morphological characteristics of the strain, and part of physiological and biochemical characteristics of the strain are researched.

1.4.2 growth characteristics of the Strain

Inoculating the strain seed liquid obtained by screening into a beef extract peptone culture medium, changing a certain condition, keeping other conditions unchanged, and measuring the OD600 value of the bacterial liquid to study the influence of temperature, pH, NaCl mass concentration and liquid loading amount on the growth of the strain.

1.4.316 Sr DNA identification

The genomic DNA of the high-efficiency high-temperature degradation bacteria obtained by screening is used as a template, and the universal primers 27f and 1492r of the general bacteria are used for PCR amplification. The method comprises the following steps: ddH2O 37.5.5 μ L; 27f 0.5 μ L; 1492r0.5 μ L; 10 × Tap Buffer 5 μ L; dNTP1 μ L; template 5 μ L; tap enzyme 0.5. mu.L. The procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 10 s; the annealing temperature adopts gradient temperature, the temperature is respectively 52.1 ℃, 53.5 ℃, 54.7 ℃, 55.6 ℃, 56.9 ℃ and 58.1 ℃, and the annealing is carried out for 40 s; extending for 1.5min at 72 ℃; for 30 cycles, and after the cycle is finished, the extension is carried out for 10min at 72 ℃. And (3) sending the PCR product to a biotechnology company for sequence determination, performing Blast analysis and comparison on the obtained sequence and a sequence in a GeneBank database, and calling out a 16Sr DNA gene sequence of a related strain with the highest similarity, wherein the sequence comparison address is as follows: http:// blast.ncbi.nlm.nih.gov/blast.cgi.

1.5 construction of efficient compost composite bacterial agent BP

And (3) measuring the growth curves of 15 strains in the CICC and the enzyme production characteristics in the fermentation process, testing the antagonism between every two strains by adopting an Oxford cup bacteriostasis method, and finally selecting 8 strains with similar growth characteristics and weak antagonism to form the BP microbial inoculum. The proportion of the components is consistent with the proportion of the corresponding organic components in the compost raw materials.

1.6 Single-factor experiment for optimizing BP fermentation conditions by using composite microbial inoculum

Optimizing a fermentation medium: three commonly used complex microbial inoculum fermentation media are selected: respectively inoculating 6% seed solution to a glucose culture medium, a bran culture medium and a seed culture medium, culturing for 48h at 25 ℃ in a shaking table at 130r/min, measuring the effective viable count and the enzyme production activity, and selecting the most suitable composite microbial inoculum fermentation culture medium.

Optimizing the fermentation temperature: selecting an optimal fermentation culture medium, inoculating 6% of seed liquid, culturing in shaking tables at 20, 25 and 30 ℃ and 130r/min for 48h, measuring the effective viable count and the enzyme production activity, and selecting an optimal fermentation temperature.

And (3) optimizing the inoculation amount: selecting optimum fermentation culture medium, respectively inoculating 3%, 6% and 8% seed solution, culturing at 25 deg.C in 130r/min shaking table for 48h, measuring effective viable count and enzyme production activity, and selecting optimum inoculum size.

1.7BP bacterial agent compost application research

1.7.1 test materials

In addition to the formulation blank as required at 1.1, 2 types of biodegradable plastic were selected for a typical type of biodegradable plastic: PLA + PBAT + starch, PLA + PBAT + CaCO3, and microcrystalline cellulose (the total organic carbon content is more than 40%) is selected as a reference.

1.7.2 compost experiments

Putting compost (activated vermiculite) and reference and sample into a 3L compost container according to the mass ratio of 6:1, and performing blank, reference and test in 3 parallels respectively under the environmental conditions: the temperature is 58 ℃ and the humidity is 100 percent; the method for measuring the carbon dioxide comprises the following steps: and (3) detecting in real time by using a CO2 sensor of the biodegradable plastic biological decomposition system.

And (3) checking the data validity basis: 1. the percentage of biodegradation of the reference material after 45d exceeded 70%; 2. the relative deviation between the percentages of biological decomposition of each compost container at the end of the test does not exceed 20%; 3. the inoculum in the blank vessel produced 50mg CO2/g volatile solids (average) to 150mg CO2/g volatile solids (average) over the pre-incubation 10 d.

2 results and discussion

2.1 selection and identification of high enzyme Activity strains

15 strains with high hydrolase activity are selected from China industrial microorganism strain preservation management center and subjected to species identification, and the results are shown in Table 4.

TABLE 4 identification of the strains species

Through continuous groping, the inventor finds that the composite strains enterobacter aryabhattai, bacillus licheniformis and corynebacterium glutamicum in the five strains can obviously improve the degradation efficiency of the bioplastic, and the strong synergistic effect can be generated among the eight strains, so that the high-temperature stage can be quickly started in a short fermentation time, the fermentation time is shortened, and the fact that the strains can generate high-activity enzymes which are most beneficial to the degradation of the bioplastic after interaction can be further illustrated.

2.2 construction of high-efficiency biodegradable Plastic compost bacterial agent BP

An efficient composting microbial inoculum BP was constructed according to the ratio shown in Table 5.

TABLE 5 compost inoculum BP composition and ratio

2.3 BP bacterial agent fermentation condition optimization

2.3.1 Medium: the proper culture medium is not only beneficial to the separation, purification and preservation of the strain, but also has great influence on the growth and metabolism of the microorganism. The influence of three different culture media, namely a seed culture medium, a bran culture medium and a glucose culture medium, on the growth and the enzyme production capacity of the thalli is experimentally researched, and the culture time is selected to be 48 hours according to the growth curve of each strain in the composite microbial inoculum. The result shows that the effective viable count of the BP microbial inoculum in a system of growing for 48 hours in three culture media is brown sugar and bran. Under the conditions of the three culture mediums, the enzyme production activity value of the microbial inoculum is higher in a seed culture medium and a erythrose culture medium, and lower in a bran culture medium. And (3) integrating the growth condition and the enzyme production capacity of the thalli, and selecting a erythrose culture medium as a BP microbial inoculum fermentation culture medium.

2.3.2 temperature: the growth process of microorganisms is also a series of enzymatic reactions, and all chemical reactions of cellular metabolism are almost catalyzed. Temperature has a significant effect on the enzymatic activity, the closer to the optimum temperature, the stronger the enzymatic activity, and the further away from the optimum temperature, the weaker the enzymatic activity, and even the loss of activity.

The effective viable count and the enzyme production capacity of the BP microbial inoculum in a system with 48h of growth at different temperatures are

25 ℃ is higher than 30 ℃ and higher than 20 ℃, and in the experiment, 25 ℃ is selected as the fermentation temperature of the BP microbial inoculum.

2.3.3 inoculum size: the inoculation is first-stage seed liquid inoculation, and the inoculation amount refers to the volume ratio of the inoculated seed liquid to the fermentation liquid. Generally, the inoculation amount is larger, so that the growth rate and the concentration of thalli can be improved, but if the inoculation amount is too large, the growth of thalli is too fast, dissolved oxygen is reduced, and the synthesis of certain fermentation products is influenced. The invention researches the influence of three different inoculation amounts on the growth and enzyme production conditions of the complex microbial inoculum, and when the inoculation amounts are respectively 3%, 6% and 8%, the larger the inoculation amount is, the higher the effective viable count of the microbial inoculum is in the same culture time. With the increase of the inoculation amount, the enzyme activity of the thalli is also increased. Therefore, 8% inoculum size was selected for this experiment for subsequent experiments.

2.4 compost test results

According to the requirements of GB/T19277.1-2011 on experimental environment, the temperature is 58 ℃, the humidity is 100%, and the pH range is 6-8 in a composting test, a CO2 infrared sensor is adopted to detect and calculate the accumulated release amount in real time. The material has the advantages that 114 days are required for the biological decomposition rate of PLA + PBAT + starch to reach 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum, 113 days are required for the biological decomposition rate of PLA + PBAT + CaCO3 to reach 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum, and the material has excellent degradation effect.

Example 2

Different from the embodiment 1, the composite microbial inoculum comprises the following strains of mass components: bacillus amyloliquefaciens: 35%, bacillus brevis: 15%, bacillus cereus: 12%, rhodopseudomonas palustris: 14%, lysine bacillus sphaericus: 15%, enterobacter aryabhattai: 8%, bacillus licheniformis: 8%, corynebacterium glutamicum: 8 percent.

The material is obtained according to the same composting experimental conditions, wherein 117 days are needed for the biological decomposition rate of PLA + PBAT + starch to reach 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum, and 115 days are needed for the biological decomposition rate of PLA + PBAT + CaCO3 to reach 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum.

Example 3

Kitchen waste degradation effect verification

Removing large-volume substances such as bones in the kitchen waste, placing the kitchen waste in a kitchen waste degradation processor, uniformly adding the composite microbial inoculum on the surface of the kitchen waste, and then fully stirring for 20min, wherein the mass ratio of the added kitchen waste to the composite microbial inoculum is 500: 1 (the total amount of the two is 3kg), carrying out fermentation treatment, starting to operate for two days, then adding 0.5kg of kitchen garbage every day, continuously operating for three months, measuring the degradation rate, wherein the degradation rate calculation formula is as follows:

C＝(A-B)/D

wherein C is the degradation rate of the kitchen waste, A is the total mass of the untreated kitchen waste and the degradation processor, B is the total mass of the degraded kitchen waste and the degradation processor, and D is the total weight of the input kitchen waste. The degradation rate after 3 days can reach 97.6 percent through verification. The composite microbial inoculum can realize the rapid and effective degradation of kitchen garbage.

Example 4

Verification of bacteriostatic effect

Adding the composite microbial inoculum into wet tissue liquid, wherein the mass ratio of the wet tissue liquid to the composite microbial inoculum is 1000: 5, the wet tissue liquid stored at the high temperature of 50 ℃ for one month and stored at the room temperature for three months is detected according to GB 15979-2002 sanitary Standard for Disposable sanitary articles, and the antibacterial effects of the wet tissue liquid on staphylococcus aureus and candida albicans reach 96.8% and 97.2% respectively according to the verification, so that the wet tissue liquid has excellent antibacterial performance.

Comparative example 1

Different from the embodiment 1, the microbial inoculum only comprises bacillus amyloliquefaciens and bacillus brevis, and is obtained according to the same composting experimental conditions, wherein the materials are characterized in that 169 days are needed when the biological decomposition rate of PLA + PBAT + starch reaches 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum, 167 days are needed when the biological decomposition rate of PLA + PBAT + CaCO3 reaches 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum, and the degradation efficiency is not high.

Comparative example 2

Compared with the embodiment 1, the microbial inoculum only comprises bacillus amyloliquefaciens, bacillus brevis and rhodopseudomonas palustris, and is obtained according to the same composting experimental conditions, wherein the materials comprise (i) PLA + PBAT + starch which can reach 90% (standard requirement) of biological decomposition rate for 153 days under the condition of the high-efficiency BP microbial inoculum, and (ii) PLA + PBAT + CaCO3 which can reach 90% (standard requirement) of biological decomposition rate for 152 days under the condition of the high-efficiency BP microbial inoculum, and the degradation efficiency is slightly improved compared with that of the comparative example 1.

Comparative example 3

Different from the embodiment 1, strains including enterobacter aryabhattai, bacillus licheniformis and corynebacterium glutamicum are omitted, and the materials are obtained according to the same composting experimental conditions, wherein the materials comprise (i) 148 days for 90% (standard requirement) of the PLA + PBAT + starch under the condition of the high-efficiency BP bacterial agent, and (ii) 147 days for 90% (standard requirement) of the PLA + PBAT + CaCO3 under the condition of the high-efficiency BP bacterial agent, and the degradation time is increased by 30%, so that the three strains play an important role in composting fermentation.

Comparative example 4

Different from the embodiment 1, the strain corynebacterium glutamicum is omitted, and the strain corynebacterium glutamicum is obtained according to the same composting experimental conditions, wherein the material has the advantages that the biodegradation rate of PLA + PBAT + starch reaching 90% (standard requirement) under the condition of the high-efficiency BP bacterial agent needs 132 days, and the biodegradation rate of PLA + PBAT + CaCO3 reaching 90% (standard requirement) under the condition of the high-efficiency BP bacterial agent needs 130 days, so that the strain corynebacterium glutamicum can play a more key role in improving the degradation rate compared with enterobacter aryabhattai and bacillus licheniformis.

Comparative example 5

Different from the embodiment 1, the corynebacterium glutamicum is replaced by Aspergillus niger (Aspergillus niger) with a CICC strain number of CICC 2115, and the corynebacterium glutamicum is obtained according to the same composting experimental conditions, wherein the material has the advantages that the biodegradation rate of PLA + PBAT + starch reaches 90% (standard requirement) under the condition of the high-efficiency BP bacterial agent needs 143 days, and the biodegradation rate of PLA + PBAT + CaCO3 reaches 90% (standard requirement) under the condition of the high-efficiency BP bacterial agent needs 144 days, which indicates that the composite strains which are not combined randomly can achieve excellent degradation effect, and depends on whether the effective synergistic effect of the degraded biological plastics can be generated among the strains.

Comparative example 6

Different from the embodiment 1, the composite microbial inoculum comprises the following strains of mass components: bacillus amyloliquefaciens: 40%, Bacillus brevis: 18%, bacillus cereus: 15%, rhodopseudomonas palustris: 20%, lysine bacillus sphaericus: 20%, enterobacter aryabhattai: 2%, bacillus licheniformis: 2%, corynebacterium glutamicum: 3 percent.

The material is obtained according to the same composting experimental conditions, wherein the biological decomposition rate of the PLA + PBAT + starch to 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum needs 152 days, and the biological decomposition rate of the PLA + PBAT + CaCO3 to 90% (standard requirement) under the condition of the high-efficiency BP microbial inoculum needs 150 days. The above results directly reflect that the content of the strain has a direct and important influence on the final degradation effect.

Comparative example 7

The culture microbial inoculum and a commercial microbial inoculum (a microbial inoculum special for an organic fertilizer) are subjected to contrast test on two biodegradable materials. The carbon dioxide accumulative release curve and the biological decomposition rate curve of the reference sample and the sample are respectively obtained, the curves at the upper part in the graphs are all the curves of overlapping cellulose 1, cellulose 2 and cellulose 3, the curves at the lower part are all the curves of overlapping the sample 1, the sample 2 and the sample 3, and the carbon dioxide release curve and the biological decomposition rate curve are basically overlapped, so that the test parallelism is proved to be good. It can be seen that the relative biological decomposition rates of two typical biodegradable plastic products (i) and (ii) in an activated vermiculite composting environment prepared from a special microbial inoculum for a commercial organic fertilizer (90 percent) respectively require 147 days and 151 days, while the biological decomposition rates of the special microbial inoculum for the efficient biodegradable plastic obtained by compounding (90 percent) under the same conditions respectively require 114 days and 113 days, so that the composting time of 1 month is shortened, and the degradation efficiency is remarkably improved.

3 conclusion

The invention screens out the high-efficiency compound microbial inoculum for composting suitable for biodegradable plastics, optimizes the proportion of the compound microbial inoculum through a biological decomposition rate test, greatly shortens the degradation time and provides data support for the standardized popularization among laboratories.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-efficiency compost composite bacterial agent is characterized in that: including Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Brevibacillus paraabrrevis), Bacillus cereus (Bacillus cereus), Rhodopseudomonas palustris (Rhodopseudomonas palustris), and Bacillus sphaericus (Lysinibacillus sphaericus).

2. The high-efficiency composting microbial inoculum of claim 1, which is characterized in that: the high-efficiency composting composite microbial inoculum also comprises Enterobacter aryabhattai (Enterobacter albureae) and Bacillus licheniformis (Bacillus licheniformis).

3. The high-efficiency composting microbial inoculum of claim 2, which is characterized in that: the high-efficiency compost composite microbial inoculum also comprises Corynebacterium glutamicum (Corynebacterium glutamicum).

4. The high-efficiency composting microbial inoculum of claim 3, which is characterized in that: bacillus amyloliquefaciens strain number CICC 10081, Bacillus parapsilosis strain number CICC 10343, Bacillus cereus strain number CICC 10041, Rhodopseudomonas palustris strain number CICC 23812, Lysinibacillus sphaericus strain number CICC 23697, Enterobacter aryabhattai strain number CICC 10013, Bacillus licheniformis strain number CICC 10092 and Corynebacterium glutamicum strain number CICC 10058 are purchased from China industrial microorganism strain preservation management center.

5. The high-efficiency composting microbial inoculum of claim 3, which is characterized in that: comprises the following strains by mass: bacillus amyloliquefaciens (Bacillus amyloliquefaciens): 22% -35%, bacillus brevis (Brevibacillus parabrevibensis): 7% -15%, Bacillus cereus (Bacillus cereus): 8% -12%, Rhodopseudomonas palustris (Rhodopseudomonas palustris): 10% -15%, lysine bacillus sphaericus (Lysinibacillus sphaericus): 10% -15%;

Or, further comprises Enterobacter aryabhattai (Enterobacter antiburiae): 7% -14%, Bacillus licheniformis (Bacillus licheniformis): 7 to 14 percent of the total weight of the mixture,

or, further comprises Corynebacterium glutamicum (Corynebacterium glutamicum): 7 to 14 percent.

6. The preparation method of the high-efficiency compost complex microbial inoculum of any one of claims 1 to 5, which is characterized by comprising the following steps: the method comprises the following steps: preparing a culture medium, inoculating the strain with the content range into the culture medium, standing at 20-37 ℃, and performing expanded culture of the strain; the culture medium is a brown sugar culture medium and comprises 3.6g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium chloride, 0.2g of magnesium sulfate heptahydrate, 4 mu M of ferric chloride and 20g of brown sugar, and the culture medium is sterilized for 20min at 121 ℃; the inoculation amount is 3% -10%.

7. The use method of the high-efficiency compost complex microbial inoculum as claimed in any one of claims 1 to 5, characterized in that: the method comprises the following steps: adding a biodegradable plastic product into a composting environment, adding a composite microbial inoculum subjected to expanded culture to form a mixed material to be treated, and performing a composting test; the composting test conditions are as follows: the temperature is 58 ℃, the humidity is 100%, the pH value is 6-8, and the composting environment is an activated vermiculite composting environment.

8. The application of the high-efficiency compost complex microbial inoculum as defined in any one of claims 1-5 and/or the preparation method as defined in claim 6 and/or the use method as defined in claim 7 in degrading biological plastics.

9. The application of the high-efficiency compost composite microbial inoculum as defined in any one of claims 1-5 in degrading household organic garbage.

10. The use of the high efficiency composting complex microbial inoculum of any one of claims 1-5 in daily chemical products.