CN111440749A - Pseudomonas pseudoalcaligenes sp.H3, screening method and application - Google Patents
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Abstract
The invention discloses Pseudomonas pseudoalcaligenes sp.H3, a screening method and application, wherein the Pseudomonas pseudoalcaligenes sp.H3 is preserved in 23.04.2020, with the preservation number of CGMCC No.19713, the Pseudomonas pseudoalcaligenes can efficiently synthesize PHA by using kitchen waste oil as a substrate, the PHA synthesis efficiency in batch experiments can reach 65%, the PHA yield is 2.6 g/L, the PHA synthesis efficiency in feeding experiments can reach 53%, and the yield can reach 8.6 g/L.
Description
Technical Field
The invention relates to the field of microbial strains, in particular to Pseudomonas pseudoalcaligenes sp.H3, a screening method and application.
Background
Polyhydroxyalkanoate (PHA) is produced by metabolic conversion of carbon sources in microbial cells under nitrogen, phosphorus or sulfur-limited conditions, and is a completely biodegradable high molecular weight bio-polyester which does not produce any toxic compounds during degradation. The PHA has similar material properties with the traditional chemical synthetic plastics, has the advantages of material variability, nonlinear optical performance, piezoelectric performance, gas barrier performance, thermoplasticity, biodegradability, good biocompatibility and the like, and is considered as an ideal substitute for the petroleum industry plastics. However, the high production cost limits the industrial production and large-scale application of PHA. A great deal of research finds that the nature has abundant microbial species and PHA can be synthesized by fermentation. However, in the fermentation process of the microorganisms, the consumption of the substrate is relatively high, and the production cost of PHA can be reduced to a great extent by selecting the substrate with low price. The kitchen waste oil generally refers to waste cooking oil, hogwash oil, waste frying oil, waste animal oil and the like, has the advantages of low market value, high carbon content and the like, and can be used as an effective carbon source for microbial culture. The kitchen waste oil is used as a synthetic substrate of PHA, and the production cost can be reduced by at least 50%. On the other hand, microorganisms capable of synthesizing PHA by using the kitchen waste oil are screened from the kitchen waste oil polluted medium, so that high value-added utilization of the kitchen waste oil can be realized, and the production cost of PHA is greatly degraded.
Upon search, the prior art has published protocols for microbial PHA synthesis. Chinese patent application CN110438030A discloses Pseudomonas putida WP07, a preparation method and application thereof. The invention can convert the swill oil into the biodegradable plastic PHA with environmental affinity by a microbiological method, can realize the resource utilization and harmless treatment of the swill oil, and simultaneously reduces the preparation cost of the PHA. However, the strain of the invention is screened for oil-contaminated soil, and although the swill oil can be used for preparing PHA, the PHA yield is too low. Chinese patent application CN 102206596A discloses a polyhydroxyalkanoate synthesis strain and a fermentation culture method thereof. The strain can synthesize PHA with short chains and medium-long chains copolymerized by taking glucose as a single carbon source, and the PHA is composed of a plurality of short chain monomers and medium-long chain monomers and has more excellent mechanical and processing properties. However, the synthesis of PHA by using the kitchen waste oil as a carbon source is not mentioned, and the synthesis efficiency of PHA is not mentioned.
Disclosure of Invention
The invention aims to provide Pseudomonas pseudomoninas sp.H3 for synthesizing polyhydroxyalkanoate by utilizing kitchen waste oil.
In order to achieve the aim, the invention provides Pseudomonas alcaligenes sp.H3, which is characterized in that the preservation number of the Pseudomonas alcaligenes sp.H3 is CGMCC No. 19713.
The invention also provides a screening method of the Pseudomonas pseudoalcaligenes sp.H3, which is characterized by comprising the following steps,
enrichment and domestication, namely adding a sample polluted by oil into an inorganic salt culture medium 1 containing kitchen waste oil, preferably with the content of the kitchen waste oil being 50 g/L, culturing in a thermostat for 5-10 days at 30 ℃, transferring a culture solution of 5-20m L into a fresh inorganic salt culture medium 1 containing the kitchen waste oil, culturing in the thermostat, continuing the previous operation, and continuing the transfer culture, wherein the kitchen oil content in the fresh inorganic salt culture medium 1 containing the kitchen waste oil used in each transfer is higher than that in the previous culture medium until the content of the kitchen waste oil reaches 100 g/L;
separation and purification: the culture solution obtained above was diluted 10 with physiological saline4-6Coating the strain on a screening culture medium containing oil, culturing in a constant temperature incubator, preferably at 25-30 ℃ and 150-200rpm for 24-72h, observing the plate in the dark by using a 365nm ultraviolet lamp, selecting a strain which emits fluorescence, and scribing the strain on an L B culture medium to obtain a grown strain, namely Pseudomonas pseudomonads sp.H3;
the inorganic salt culture medium 1 comprises the following components: (NH)4)2SO4:1g/L；KH2PO4:1g/L；Na2HPO4·12H2O:11.1g/L；MgSO40.2 g/L, trace element liquid (FeCl)3:9.7g/L；CaCl2:7.8g/L；CuSO4·5H2O:0.156g/L；CoCl2：0.119g/L；NiCl2:0.118g/L；CrCl20.062 g/L dissolved in 0.1M hydrochloric acid) 1M L/L.
The screening culture medium is obtained by adding 0.5 mu g/m L Nile blue solution, 10-30 g/L of kitchen waste oil and fat and 2% agar powder into an inorganic salt culture medium 1, sterilizing at 121 ℃ for 21min, cooling and solidifying, wherein the Nile blue solution is obtained by dissolving 1g Nile blue into 100m L dimethyl sulfoxide, and obtaining the Nile blue solution after full dissolution.
Further, the kitchen waste oil is frying waste oil, cooking waste oil, hogwash oil, mixed waste oil obtained by separating kitchen waste after treatment, waste oil extracted from plants and animals or mixture of any two or more of the above.
The invention also provides application of the Pseudomonas pseudoalcaligenes sp.H3.
Further, the application refers to the application of synthesizing the polyhydroxy fatty acid ester from the kitchen waste oil.
Further, the kitchen waste oil is frying waste oil, cooking waste oil, hogwash oil, mixed waste oil obtained by separating kitchen waste after treatment, waste oil extracted from plants and animals or mixture of any two or more of the above;
optionally, the structural monomer of the polyhydroxyalkanoate comprises C8，C10And C12Monomeric 3-hydroxy fatty acid esters.
The invention provides a method for synthesizing polyhydroxyalkanoate by utilizing Pseudomonas pseudoalcaligenes sp.H3, which is characterized by comprising the following steps,
preparing a seed solution and a bacterial suspension, namely inoculating Pseudomonas pseudoalcaligenes sp.H3 into L B culture medium to be cultured to logarithmic phase, preferably obtaining the seed solution after 12-16h, and preparing bacterial cells obtained by centrifuging the seed solution into the bacterial suspension with OD600 of 1.0-2.0 by adding physiological saline;
inoculating, namely adding 10-50 g/L of waste kitchen oil and fat into an inorganic salt culture medium 2, inoculating 1-5% of the obtained bacterial suspension, and then performing shake culture, preferably performing shake culture at 30 ℃, 150-;
and (3) separating and purifying thalli: centrifuging the obtained bacteria to obtain bacteria, adding deionized water or ultrapure water to dilute by 10-15 times, mixing thoroughly, adding n-hexane with the same volume as the bacteria liquid to be diluted to remove impurities and grease, centrifuging, and freeze-drying to obtain dry bacteria;
extracting polyhydroxyalkanoate, namely adding chloroform into the obtained dry thalli, preferably, the adding amount of the chloroform is 50-100m L/g dry thalli, carrying out ultrasonic treatment to break cells, preferably, carrying out ultrasonic treatment for 10-30min, then fully heating and stirring for 3-6h, preferably 4h at 80-100 ℃ and preferably 90 ℃ by using a water bath magnetic stirrer, and centrifuging to remove mushroom dregs to obtain polyhydroxyalkanoate of a chloroform phase primarily;
purification of polyhydroxyalkanoate: adding 2-5% of deionized water or ultrapure water into the chloroform-phase polyhydroxyalkanoate, fully shaking and washing, centrifuging to obtain pure chloroform-phase polyhydroxyalkanoate, finally pouring the pure chloroform-phase polyhydroxyalkanoate into a culture dish in a fume hood, and completely volatilizing chloroform to obtain purified polyhydroxyalkanoate;
the inorganic salt culture medium 2 comprises the following components: KH (Perkin Elmer)2PO4:1.5g/L；Na2HPO4·12H2O:8.95g/L；MgSO4:0.098g/L；CaCl20.0076 g/L, 1m L/L trace element liquid (H)3BO3：0.3g/L；CoCl2·6H2O:0.2g/L；MnCl2·4H2O:0.03g/L；NaMO4·2H2O:0.03g/L；NiCl2·6H2O:0.02g/L；CuSO4·5H2O:0.01g/L；ZnCl20.047 g/L), and the nitrogen source is 0.32g N/L.
Further, the nitrogen source may be urea, NH4Cl，(NH4)2SO4，NH4NO3。
Further, the kitchen waste oil is frying waste oil, cooking waste oil, hogwash oil, mixed waste oil obtained by separating kitchen waste after treatment, waste oil extracted from plants and animals, or mixture of any two or more of the above.
Further, the structural monomer of the polyhydroxyalkanoate comprises C8，C10And C12Monomeric 3-hydroxy fatty acid esters.
The Pseudomonas pseudomonads sp.H3 for producing the alkali is a new strain which is screened from the nature and can synthesize medium-long-chain polyhydroxyalkanoate by using kitchen waste oil as a substrate, although Pseudomonas bacteria can utilize various carbon sources as the substrate to synthesize PHA, wild strains which use the kitchen waste oil as the substrate and have higher synthesis efficiency are not reported, the efficiency of synthesizing PHA by using oil by using common wild Pseudomonas is about 10-20 percent, while the wild Pseudomonas pseudomonads sp.H3 obtained by domestication and screening of the invention can efficiently synthesize PHA by using the kitchen waste oil as the substrate, the synthesis efficiency of PHA in batch experiments can reach 65 percent, the PHA yield is 2.6 g/L, the synthesis efficiency of PHA in feed experiments can reach 53 percent, and the yield can reach 8.6 g/L.
Drawings
FIG. 1 is a scanning electron micrograph of Pseudomonas sp.H 3.
Figure 2 is a phylogenetic tree diagram of Pseudomonas sp.h 3.
FIG. 3 is a graph showing the cell yield and PHA yield of the strain Pseudomonas sp.H3 under the initial pH conditions of different media in example 3.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The media in the following examples are:
the inorganic salt culture medium 1 comprises the following components: (NH)4)2SO4:1g/L；KH2PO4:1g/L；Na2HPO4·12H2O:11.1g/L；MgSO40.2 g/L, trace element liquid (FeCl)3:9.7g/L；CaCl2:7.8g/L；CuSO4·5H2O:0.156g/L；CoCl2：0.119g/L；NiCl2:0.118g/L；CrCl20.062 g/L dissolved in 0.1M hydrochloric acid) 1M L/L.
The screening culture medium comprises inorganic salt culture medium 1, Nile blue solution (final concentration of 0.5 μ g/m L), 20 g/L of waste kitchen oil and fat, and 2% agar powder, wherein the Nile blue solution is obtained by dissolving 1g of Nile blue in 100m L dimethyl sulfoxide, and the culture medium is used after being sterilized at 121 ℃ for 21 min.
The preparation method of the inorganic salt culture medium 2 comprises the following steps: KH (Perkin Elmer)2PO4:1.5g/L；Na2HPO4·12H2O:8.95g/L；MgSO4:0.098g/L；CaCl20.0076 g/L, 1m L/L trace element liquid (H)3BO3：0.3g/L；CoCl2·6H2O:0.2g/L；MnCl2·4H2O:0.03g/L；NaMO4·2H2O:0.03g/L；NiCl2·6H2O:0.02g/L；CuSO4·5H2O:0.01g/L；ZnCl20.047 g/L), nitrogen source (NH)4)2SO4:1.5g/L。
L B medium comprises tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L), and 2% agar powder can also be added to make L B solid medium.
Example 1: screening, domestication, separation and identification of strain Pseudomonas sp.H3
Weighing 1g of greasy dirt and dust on a range hood, placing the greasy dirt and dust in a conical flask with the length of 250m L, adding the greasy dirt and dust in an oil-containing inorganic salt culture medium 1 with the length of 100m L, adding the kitchen waste oil and dust in the oil-containing inorganic salt culture medium 1 with the addition of 50 g/L, placing the mixture in a constant-temperature shaking table with the temperature of 30 ℃ and the rpm of 160 for culture, taking the culture solution with the length of 20m L from the mixture after 5 days, transferring the culture solution with the length of 20m L into a fresh inorganic salt culture medium, increasing the kitchen waste oil and dust addition to 70 g/L, and continuing to culture and transfer according to the operation steps until the kitchen waste oil and dust content in the inorganic salt culture medium is 100 g/L.
The resulting dilution 105The bacterial strain is coated on a screening culture medium, the screening culture medium is placed in an incubator at 30 ℃ for culturing for 72h, a plate is observed by utilizing a 365nm ultraviolet lamp, a fluorescent strain is selected and named as Pseudomonas alcaligenes sp.H3, the strain Pseudomonas sp.H3 is cultured on an L B solid culture medium for 24-36h, the bacterial colony is circular and faint yellow, the surface is smooth, the bacterium is judged to be a gram-negative bacterium by a gram staining method, and the single bacterium is observed to be rod-shaped by a scanning electron microscope, wherein the result is shown in figure 1.
The kit is used for extracting genome DNA of the strain H3, and the strain H3 obtained by separation is classified and identified by a 16s rRNA gene sequence method. The result of comparison of the measured 16s rRNA gene sequence with the sequence reported in GenBank shows that the homology of the strain H3 with Pseudomonas alcaligenes reaches 98.84 percent, which indicates that the strain H3 is a Pseudomonas (Pseudomonas), in particular to an alcaligenes. The phylogenetic tree of Pseudomonas sp.H3 is shown in FIG. 2. The strain is preserved in China general microbiological culture Collection center at 23.04.2020, with the following preservation information:
the strain name: pseudomonas pseudoalcaligenes sp.H3;
the preservation date is as follows: 23/04/2020;
the preservation unit: west road No.1 hospital No. 3, north chen of chaoyang district, beijing, china general microbiological culture collection center (CGMCC);
the preservation number is: CGMCC No. 19713.
Example 2: synthesis of polyhydroxyalkanoate by strain Pseudomonas sp.H3 under different pH values
Inoculating the strain Pseudomonas sp.H3 into L B culture medium, culturing for 14h to obtain seed liquid, and adding physiological saline to obtain thallus to obtain bacterial suspension with OD600 of 1.0.
Placing 100m L inorganic salt culture medium 2 into a 250m L conical flask, adjusting the initial pH values to 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0 respectively, adding 20 g/L kitchen waste oil and fat respectively, sterilizing the inorganic salt culture medium 2 with different pH values at 121 ℃ in an autoclave for 21min, respectively inoculating 4% of bacterial suspension into the sterilized inorganic salt culture medium 2, placing the inorganic salt culture medium 2 into a 30 ℃ shaking table for culturing for 72h at 200rpm, centrifugally collecting thalli, adding deionized water for diluting by 15 times, adding n-hexane with the volume equal to that of a diluent for washing to remove oil and fat adsorbed by the thalli, finally freeze-drying the thalli obtained by centrifugation, placing the dried thalli into the conical flask, adding chloroform with the mass 50 times of the mass of the thalli, carrying out ultrasonic treatment for 15min, then fully heating and stirring the thalli at 90 ℃ by using a water bath magnetic stirrer for 4h, centrifugally removing bacterial slag to obtain 5% of a chloroform-phase polyhydroxyalkanoate, adding 5% of the chloroform-phase polyhydroxyalkanoate into a chloroform-phase centrifugal culture medium, placing the centrifugal culture vessel for obtaining PHA, and obtaining the yield of the PHA which is obtained by weighing the strain under the alkaline cell culture medium under the condition that the alkaline cell culture medium, wherein the pH value is equal to obtain the yield of PHA (the PHA) of the PHA 2, the PHA is obtained by the PHA, and the PHA, and the PHA is obtained by the PHA, and the yield of the PHA, the yield.
Example 3 Synthesis of polyhydroxyalkanoate by feeding and fermenting kitchen waste oil and fat with Pseudomonas sp.H3 Strain
Inoculating Pseudomonas sp.H3 into L B culture medium, culturing for 14 hr to obtain seed liquid, centrifuging to obtain thallus, adding physiological saline to obtain thallus suspension with OD600 of 2.0, inoculating the thallus suspension into 5-5L fermenter, and culturing in 3.0L inorganic salt culture medium containing KH2PO4:1.5g/L；Na2HPO4·12H2O:8.95g/L；MgSO4:0.098g/L；CaCl2:0.0076g/L1m L trace element liquid and nitrogen source (NH)4)2SO41.5 g/L of kitchen waste oil and fat, 10 g/L of pH 7.0, controlling the air flow at 1vvm, stirring the fermentation tank at the rotating speed of 300-.
Table 1 shows the experimental results of PHA synthesis by feeding fermentation of the strain Pseudomonas sp.H3 by using kitchen waste oil and fat, and the yield of the strain which can grow up to 16 g/L under the condition of feeding fermentation culture reaches 8.6 g/L.
TABLE 1 results table
Note that the dry weight of the cells (g/L) was 3.2g by 1L/0.2L was 16g, i.e., the dry weight of the cells was 16 g/L.
PHA production (g/L) ═ PHA content by dry weight of cells 16 g/L ═ 53.63% ═ 8.6 g/L.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. Pseudomonas alcaligenes sp.H3, wherein the preservation number of the Pseudomonas alcaligenes sp.H3 is CGMCC No. 19713.
2. A method for screening Pseudomonas pseudoalcaligenes sp.H3 as claimed in claim 1, comprising the steps of,
enrichment and domestication, namely adding a sample polluted by oil into an inorganic salt culture medium 1 containing kitchen waste oil, preferably with the content of the kitchen waste oil being 50 g/L, culturing in a thermostat for 5-10 days at 30 ℃, transferring a culture solution of 5-20m L into a fresh inorganic salt culture medium 1 containing the kitchen waste oil, culturing in the thermostat, continuing the previous operation, and continuing the transfer culture, wherein the kitchen oil content in the fresh inorganic salt culture medium 1 containing the kitchen waste oil used in each transfer is higher than that in the previous culture medium until the content of the kitchen waste oil reaches 100 g/L;
separation and purification: the culture solution obtained above was diluted 10 with physiological saline4-6Coating the strain on a screening culture medium containing oil, culturing in a constant temperature incubator, preferably at 25-30 ℃ and 150-200rpm for 24-72h, observing the plate in the dark by using a 365nm ultraviolet lamp, selecting a strain which emits fluorescence, and scribing the strain on an L B culture medium to obtain a grown strain, namely Pseudomonas pseudomonads sp.H3;
the inorganic salt culture medium 1 comprises the following components: (NH)4)2SO4:1g/L；KH2PO4:1g/L；Na2HPO4·12H2O:11.1g/L；MgSO40.2 g/L, trace element liquid (FeCl)3:9.7g/L；CaCl2:7.8g/L；CuSO4·5H2O:0.156g/L；CoCl2：0.119g/L；NiCl2:0.118g/L；CrCl20.062 g/L dissolved in 0.1M hydrochloric acid) 1M L/L;
the screening culture medium is obtained by adding 0.5 mu g/m L Nile blue solution, 10-30 g/L of kitchen waste oil and fat and 2% agar powder into an inorganic salt culture medium 1, sterilizing at 121 ℃ for 21min, cooling and solidifying, wherein the Nile blue solution is obtained by dissolving 1g Nile blue into 100m L dimethyl sulfoxide, and obtaining the Nile blue solution after full dissolution.
3. The method for screening Pseudomonas sp.H3 as claimed in claim 2, wherein the kitchen waste oil is frying waste oil, cooking waste oil, hogwash oil, mixed waste oil separated from kitchen waste after treatment, waste oil extracted from plants and animals, or a mixture of any two or more of the above.
4. Use of Pseudomonas sp.H3 as defined in claim 1 for the production of Pseudomonas sp.H3.
5. The use according to claim 1, wherein the use refers to the use for synthesizing polyhydroxyalkanoate from kitchen waste oil.
6. The use of claim 1, wherein the kitchen waste oil is frying waste oil, cooking waste oil, hogwash oil, mixed waste oil separated after kitchen waste treatment, waste oil extracted from plants and animals, or a mixture of any two or more of the above;
optionally, the structural monomer of the polyhydroxyalkanoate comprises C8，C10And C12Monomeric 3-hydroxy fatty acid esters.
7. A process for the synthesis of polyhydroxyalkanoates using Pseudomonas sp.H3 as described in claim 1, comprising the steps of,
preparing a seed solution and a bacterial suspension, namely inoculating Pseudomonas pseudoalcaligenes sp.H3 into L B culture medium to be cultured to logarithmic phase, preferably obtaining the seed solution after 12-16h, and preparing bacterial cells obtained by centrifuging the seed solution into the bacterial suspension with OD600 of 1.0-2.0 by adding physiological saline;
inoculating, namely adding 10-50 g/L of waste kitchen oil and fat into an inorganic salt culture medium 2, inoculating 1-5% of the obtained bacterial suspension, and then performing shake culture, preferably performing shake culture at 30 ℃, 150-;
and (3) separating and purifying thalli: centrifuging the obtained bacteria to obtain bacteria, adding deionized water or ultrapure water to dilute by 10-15 times, mixing thoroughly, adding n-hexane with the same volume as the bacteria liquid to be diluted to remove impurities and grease, centrifuging, and freeze-drying to obtain dry bacteria;
extracting polyhydroxyalkanoate, namely adding chloroform into the obtained dry thalli, preferably, the adding amount of the chloroform is 50-100m L/g dry thalli, carrying out ultrasonic treatment to break cells, preferably, carrying out ultrasonic treatment for 10-30min, then fully heating and stirring for 3-6h, preferably 4h at 80-100 ℃ and preferably 90 ℃ by using a water bath magnetic stirrer, and centrifuging to remove mushroom dregs to obtain polyhydroxyalkanoate of a chloroform phase primarily;
purification of polyhydroxyalkanoate: adding 2-5% of deionized water or ultrapure water into the chloroform-phase polyhydroxyalkanoate, fully shaking and washing, centrifuging to obtain pure chloroform-phase polyhydroxyalkanoate, finally pouring the pure chloroform-phase polyhydroxyalkanoate into a culture dish in a fume hood, and completely volatilizing chloroform to obtain purified polyhydroxyalkanoate;
the inorganic salt culture medium 2 comprises the following components: KH (Perkin Elmer)2PO4:1.5g/L；Na2HPO4·12H2O:8.95g/L；MgSO4:0.098g/L；CaCl20.0076 g/L, 1m L/L trace element liquid (H)3BO3：0.3g/L；CoCl2·6H2O:0.2g/L；MnCl2·4H2O:0.03g/L；NaMO4·2H2O:0.03g/L；NiCl2·6H2O:0.02g/L；CuSO4·5H2O:0.01g/L；ZnCl20.047 g/L), and the nitrogen source is 0.32g N/L.
8. The method of claim 7, wherein the nitrogen source is urea, NH4Cl，(NH4)2SO4，NH4NO3。
9. The method according to claim 7, wherein the kitchen waste oil is frying waste oil, cooking waste oil, waste oil from waste cooking, hogwash oil, mixed waste oil separated from kitchen waste after treatment, waste oil extracted from plants and animals, or a mixture of any two or more of the above.
10. The method of claim 7, wherein the polyhydroxyalkanoate comprises C as a structural monomer8，C10And C12Monomeric 3-hydroxy fatty acid esters.
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| CN111979158A (en) * | 2020-09-03 | 2020-11-24 | 广州希奕餐厨降解设备有限公司 | Compound microbial inoculum for food waste compost as well as preparation method and application thereof |
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| CN111979158A (en) * | 2020-09-03 | 2020-11-24 | 广州希奕餐厨降解设备有限公司 | Compound microbial inoculum for food waste compost as well as preparation method and application thereof |
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