CN115058357A - Bacterial strain for biological drying of kitchen waste, screening method and application - Google Patents
Bacterial strain for biological drying of kitchen waste, screening method and application Download PDFInfo
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Abstract
The invention discloses a strain for biological drying of kitchen waste, a screening method and application, and relates to the field of biological drying of kitchen waste, wherein the strain is Bacillus subtilis or Bacillus amyloliquefaciens; the bacillus subtilis (Bacillus subtilis) is named as FW91 and has a preservation number of: CGMCC No. 23139; the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is named as FW1 and has the preservation number as follows: CGMCC No. 23138. The strain screened by the invention is cooperatively used in the biochemical drying treatment of the kitchen waste, and is mixed with the sawdust and the kitchen waste according to a certain proportion, so that the removal efficiency of CODcr in the kitchen waste, particularly the removal rate of starch is up to more than 88%, and a large amount of biological heat is generated, so that the water in the kitchen waste is evaporated, the solid-liquid separation is realized, the amount of the kitchen waste is rapidly reduced, and the treatment and transportation cost of the kitchen waste is reduced.
Description
Technical Field
The invention relates to the field of biological drying of kitchen waste, in particular to a strain for biological drying of kitchen waste, a screening method and application.
Background
Kitchen waste mainly comes from residential houses, commercial institutions (such as restaurants), social institutions (such as school canteens), factory restaurants and the like, and the problem of kitchen waste treatment and disposal becomes a potential hazard source influencing food safety and ecological safety. In recent years, the production amount of kitchen waste is rapidly increased. As a big catering country in China, the annual yield of the catering garbage in China in 2017 reaches 6.3 multiplied by 10 7 t (Schbo, et al, 2019), is expected to grow to 4.80X 10 by 2030 8 t (XU Q Y, et al.2011). Such a huge amount of kitchen waste has become an important problem restricting the development of urban economy and the construction of social civilization.
The kitchen waste mainly comprises: food residues of restaurants, canteens and the like, processing wastes of fruits, vegetables, meat, grease, pastries and the like, and expired foods and the like. The kitchen waste has the characteristics of high yield, dispersed sources, easy decay and the like, so the requirements for collection, transportation and disposal of the kitchen waste are higher, and the kitchen waste needs to be timely, safe and sanitary. In recent years, with the continuous promotion of municipal waste classification work, in order to better collect and dispose of kitchen waste in a centralized manner, municipal kitchen waste collection and transportation systems face a plurality of problems of large construction investment, high operation cost, large management difficulty and the like.
The kitchen waste has the characteristics of high organic matter content, high water content, high salinity, low calorific value, easy biodegradation and the like. The kitchen waste treatment and disposal technology mainly comprises the following steps: anaerobic fermentation, aerobic composting, feed production, incineration, landfill and the like are the most common (Lufan, et al, 2017), but anaerobic fermentation is the most common, but the biogas residue and biogas slurry obtained by anaerobic fermentation and organic fertilizers obtained by aerobic composting have the problem of secondary pollution, if a farmland absorption mode is adopted to treat biogas residue, the risk of soil salinization exists, and the fertilizers obtained by aerobic composting have the defects of poor fertilizer efficiency, low farmer acceptance, seasonal agricultural fertilizer and other factors, so that the implementation of the method is limited. Other ways, such as: landfill is gradually prohibited by law, feed is a food safety problem, and high water content and low calorific value make incineration treatment expensive. The existing kitchen waste treatment and disposal modes have obvious defects and shortcomings.
Therefore, it is necessary to develop a novel treatment process with low running cost, simple operation and high treatment efficiency. With the development of organic solid waste treatment technology, biological drying technology is gradually developed in recent years, and as a reduction treatment technology for high-water-content organic solid waste, the process utilizes the heat generated by the metabolic activity of aerobic microorganisms to assist ventilation to improve the dehydration rate of materials, thereby reducing the moisture content of waste and achieving the purpose of reduction. In addition, the kitchen waste is rich in carbohydrate, protein, fat, nitrogen, phosphorus, calcium, potassium and other nutrient elements, has high C/N and is easy to degrade by aerobic microorganisms. Practice proves that the method for decomposing and treating the kitchen waste by utilizing the microorganism strengthening technology can obviously improve the degradation efficiency of organic pollutants and the generation efficiency of biological heat in unit time, is a small-investment and high-efficiency method, and is a measure for inoculating exogenous high-efficiency microorganisms, adding auxiliary material matrix for fermentation and the like. The microbial preparation can be rapidly fermented, efficiently degrade organic matters and rapidly remove water. It has the advantages of high efficiency, low cost and the like. In view of the above, the application provides a strain for biological drying of kitchen waste, a screening method and an application.
Disclosure of Invention
The invention aims to solve the technical problem of providing a strain for biological drying of kitchen waste, a screening method and application. The aim of biological drying treatment of the kitchen waste is quickly and efficiently achieved.
In order to solve the technical problems, the first object of the present invention is to provide a strain for biological drying of kitchen waste, wherein the strain is Bacillus subtilis or Bacillus amyloliquefaciens; has been preserved in China general microbiological culture Collection center (CGMCC) at 8 months and 12 days in 2021; the Bacillus subtilis (Bacillus subtilis) is named as FW91 and has a preservation number of: CGMCC No. 23139; the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is named as FW1 and has the preservation number as follows: CGMCC No. 23138.
The beneficial effects of the invention are: the strain screened by the invention is cooperatively used in biochemical drying treatment of the kitchen waste, and is mixed with the wood chips and the kitchen waste according to a certain proportion, so that the removal efficiency of CODcr in the kitchen waste, particularly the removal rate of starch is up to more than 88%, a large amount of biological heat is generated, the water in the kitchen waste is evaporated, solid-liquid separation is realized, the kitchen waste is reduced rapidly, the treatment and transportation cost of the kitchen waste is reduced, and the residual residues can be used as humus or organic fertilizers for agriculture and forestry planting.
The second purpose is to provide a screening method of the bacterial strain for biological drying of the kitchen waste, which comprises the following steps:
step 1: sampling and domesticating culture of kitchen waste: taking the kitchen waste and the sawdust, adjusting the carbon nitrogen ratio to be (15-35):1, fully mixing, and adjusting the water content to be 55-65% to obtain a mixed material; culturing and domesticating the mixed material by aerobic fermentation for 5-9 days, and heating the mixed material to 65-70 deg.C to obtain cultured and domesticated mixed material;
step 2: separating and screening strains: mixing the cultured and domesticated mixed material with sterile water according to the weight ratio of 1 (4-5), and repeatedly and violently shaking to form mixed bacteria liquid; diluting the mixed bacterial liquid into 10 by adopting a multiple dilution method -1 To 10 -7 Gradient bacterial suspension, namely taking the bacterial suspension with the same volume and each dilution degree to carry out scraper coating on a solid culture medium taking kitchen waste as a carbon source, observing the colony morphology in a constant-temperature incubator at 40-50 ℃, selecting a colony with good growth in the solid culture medium by comparing the colony morphology, repeatedly carrying out streak separation and purification until the colony morphology characteristics obtained each time are consistent, and obtaining three strains which are named as FW1, FW2 and FW91 respectively;
and step 3: three strains were rescreened by performance assay: respectively carrying out amplification culture on FW1, FW2 and FW91 strains to obtain an amplified FW1 microbial inoculum, an amplified FW2 microbial inoculum and an amplified FW91 microbial inoculum, respectively mixing the amplified FW1 microbial inoculum, the amplified FW1 microbial inoculum and the amplified FW91 microbial inoculum with mixed materials, and carrying out aerobic fermentation; by comparing the crude fat degradation rate, the starch degradation rate, the protein degradation rate, the SCOD removal rate and the total weight loss rate of the expanded FW1 microbial inoculum, FW2 microbial inoculum and FW91 microbial inoculum, the FW1 and FW91 bacterial strains with obvious effects, namely the bacterial strains for biological drying of the kitchen waste, are obtained.
Further, the method for expanding the strains in the step 3 comprises the following steps:
1) adjusting the pH of the culture medium to 6.5-7.5, and sterilizing with high pressure steam;
2) inoculating strains (FW1, FW2 and FW91) in a clean bench, performing shake culture at 33-40 deg.C and 100-120r/min for 36-60 hr until the culture solution of the propagation medium is completely turbid, and when the turbidity is 0.4-0.6 McLeod, the effective bacteria content of the culture solution of the propagation medium reaches 10 7 -10 9 cfu/mL, completing the strain expansion culture.
The beneficial effect of adopting the further scheme is that: can effectively screen out the strains for biological drying of the kitchen waste.
Further, the solid medium is: 20g of kitchen waste dried powder, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 0.2% of 1% ferric trichloride solution, 0.05% of ammonium sulfate, 0.05% of yeast powder, 0.05% of sodium chloride, 1.5% of agar and 1000ml of distilled water;
the culture medium for expanding culture comprises: 20g of kitchen waste dried powder, 0.05 percent of dipotassium hydrogen phosphate, 0.05 percent of magnesium sulfate, 0.2 percent of 1 percent ferric trichloride solution, 0.05 percent of ammonium sulfate, 0.05 percent of yeast powder, 0.05 percent of sodium chloride and 1000ml of distilled water.
The third purpose is to provide a composite microbial agent for biological drying of kitchen waste, the composite microbial agent comprises bacillus subtilis FW91 and bacillus amyloliquefaciens FW1, and the effective bacteria content ratio of the bacillus subtilis FW91 to the bacillus amyloliquefaciens FW1 is (1-2): (1-2).
The beneficial effect of adopting the further scheme is that: through the synergy of bacillus subtilis FW91 and bacillus amyloliquefaciens FW1, the fast fermentation of the kitchen can be realized, organic matters can be efficiently degraded, and water can be fast removed.
Further, the effective bacteria content ratio of the bacillus subtilis FW91 to the bacillus amyloliquefaciens FW1 is 1: 1.
Further, the compound microbial inoculum consists of the following components in parts by weight: bacillus subtilis FW 910.5-2%, bacillus amyloliquefaciens FW 10.5-2%, dipotassium hydrogen phosphate 0.04-0.06%, magnesium sulfate 0.04-0.07%, 1% ferric chloride solution 0.15-0.3%, ammonium sulfate 0.04-0.06%, yeast powder 0.04-0.06%, sodium chloride 0.03-0.06% and the balance of water.
Further, the compound microbial inoculum consists of the following components in parts by weight: bacillus subtilis FW 911-1.5 wt%, bacillus amyloliquefaciens FW 11-1.5 wt%, dipotassium hydrogen phosphate 0.05-0.06 wt%, magnesium sulfate 0.05-0.06 wt%, 1% ferric chloride solution 0.20-0.25 wt%, ammonium sulfate 0.04-0.05 wt%, yeast powder 0.04-0.05 wt%, sodium chloride 0.04-0.05 wt%, and water for the rest.
The beneficial effect of adopting the further scheme is that: the commercial kitchen waste treatment microbial inoculum prepared from the composite strain can obviously improve the degradation efficiency of organic pollutants, improve the generation efficiency of biological heat in unit time and reduce the treatment cost of kitchen waste.
The fourth purpose is to provide a preparation method of the composite microbial inoculum for biological drying of kitchen waste, which comprises the steps of expanding and culturing a strain of bacillus subtilis FW91 and bacillus amyloliquefaciens FW1 according to claim 3 respectively to obtain an expanded and cultured strain of bacillus subtilis FW91 and an expanded and cultured bacillus amyloliquefaciens FW1, and culturing the expanded and cultured strain of bacillus subtilis FW91 and the expanded and cultured bacillus amyloliquefaciens FW1 for 16-24 hours at 30 +/-5 ℃ and 150-200 rpm according to the weight ratio of (1-2) to obtain the composite microbial inoculum.
The fifth purpose is to provide the application of the composite microbial inoculum for the biological drying of the kitchen waste, and the composite microbial inoculum is applied to the treatment of the kitchen waste.
The beneficial effect of adopting the further scheme is that: the compound strain is prepared into a commercialized kitchen waste treatment microbial inoculum, is used as an exogenous addition strain in the field of kitchen waste treatment, reduces the kitchen waste treatment cost, and has wide application prospect.
Drawings
FIG. 1 is a diagram showing the growth morphology of Bacillus subtilis;
FIG. 2 is a gram stain of Bacillus subtilis;
FIG. 3 is a diagram showing the growth of Bacillus amyloliquefaciens;
FIG. 4 is a gram stain of Bacillus amyloliquefaciens;
FIG. 5 is a simplified diagram of a kitchen waste treatment process;
FIG. 6 is a graph showing the weight change of kitchen waste treated for 30 days in the application example of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
In the technical scheme of the invention, the reagents are purchased from biochemical shops if not specifically mentioned, and the technical scheme is conventional in the field if not specifically mentioned.
The detection method of each index in the test comprises the following steps: the chemical oxygen demand (SCODcr) is measured by a bichromic acid-base method; the determination of the water content adopts a gravimetric method; fat determination adopts 'determination of fat in food safety national standard food' GB 5009.6-2016; starch determination of starch in national food safety Standard GB 5009.9-2016; protein determination of protein in national food safety Standard GB 5009.5-2010.
The solid culture medium used in the test is 20g of kitchen waste dried powder, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 0.2% of 1% ferric chloride solution, 0.05% of ammonium sulfate, 0.05% of yeast powder, 0.05% of sodium chloride, 1.5% of agar and 1000ml of distilled water;
the culture medium for expanding culture is 20g of kitchen waste dried powder, 0.05 percent of dipotassium hydrogen phosphate, 0.05 percent of magnesium sulfate, 0.2 percent of 1 percent ferric trichloride solution, 0.05 percent of ammonium sulfate, 0.05 percent of yeast powder, 0.05 percent of sodium chloride and 1000ml of distilled water. Used for expansion culture.
Example 1: separation, purification and identification of Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1
1. Sampling and domestication culture of kitchen waste
Adjusting according to a carbon-nitrogen ratio of 25:1, taking 4kg of kitchen waste and 1.6kg of sawdust, fully mixing, adjusting the water content to 65% to obtain a mixed material, placing the mixed material in a fermentation reactor, carrying out mixed fermentation in a laboratory, stirring the mixed material 1 time every 6 hours, mixing and transferring the materials by stirring to enable all the materials to fully contact with oxygen, ventilating 10min every time by blowing, stopping 20min, circularly ventilating by blowing to ensure that the oxygen amount in a fermentation bin is enough to supply the oxygen demand of fermentation bacteria, detecting the change of the temperature, the pH, the crude fat, the starch, the salt and the organic matters of the mixed material every day, and carrying out seven-day culture and domestication, wherein the temperature of the mixed material is raised to 68 ℃;
2. isolation and screening of strains
And taking a 10g to 500mL triangular flask of the mixed material cultured and domesticated for seven days, adding about 300mL of sterile water, and repeatedly and violently shaking to form mixed bacteria liquid. Diluting to 10% by multiple dilution method -1 To 10 -7 And (3) inoculating 100 microliters of solid culture medium scraper coating taking kitchen waste as a carbon source to each dilution degree of the gradient bacterial suspension, and then inverting the culture dish to observe the colony morphology every day in a constant-temperature incubator at 45 ℃. And selecting well-grown colonies from a solid culture medium by comparing the colony morphology, carrying out streak separation and purification, inverting the culture dish, observing the colony morphology in a constant temperature incubator at 45 ℃ every day until colonies with consistent characteristics are obtained, then selecting the colonies, repeating the previous step of streak separation and purification culture until the colony morphology characteristics obtained each time are consistent, and no other mixed bacteria colonies appear, thereby obtaining three strains which are named as FW1, FW2 and FW91 respectively.
3. Preliminary determination and screening of three strains
Selecting the obtained three strains with sterile inoculating loop, inoculating to sterilized culture medium, shake culturing for a period of time until the liquid culture medium is completely turbid (effective bacteria content is 10) 8 -10 9 cfu/mL) to obtain an expanded FW1 microbial inoculum, an expanded FW2 microbial inoculum and an expanded FW91 microbial inoculum; then respectively adding the expanded FW1 microbial inoculum, the expanded FW2 microbial inoculum and the expanded FW2 microbial inoculumAdding an FW91 microbial inoculum into a mixed material of kitchen waste and sawdust (the carbon-nitrogen ratio is 25:1, the water content is 65%, and the total mass is 5.6kg), and repeating for 2-3 times as a primary determination group; the mixed material without inoculated propagation strain (carbon nitrogen ratio 25:1, water content 65%, total mass 5.6kg) was used as a blank control. Stirring the mixed materials for 1 time every 6 hours, mixing the materials by stirring and transferring mass so that all the materials are fully contacted with oxygen, ventilating for 10min every time, stopping ventilation for 20min, circularly ventilating by air blast to ensure that the oxygen amount in a fermentation bin is enough to supply the oxygen demand of zymophyte, detecting the temperature, pH, crude fat, starch, salt, organic matters and weight change of the mixed materials every day, and performing a seven-day test. Primary assay group: the temperature of the mixed material added with the expanded FW91 microbial inoculum is raised to 68 ℃, the heating rate is 68 ℃/3 days, the degradation rate of crude fat reaches 78.52%, the degradation rate of starch reaches 74.75%, the degradation rate of protein reaches 89.27%, and the removal rate of SCOD (soluble COD) reaches 79.45%; the total weight loss rate reaches 78.45 percent. The material added with the expanding culture FW2 microbial inoculum has the temperature rise rate of 52 ℃/3 days, the removal rate of crude fat reaches 40.75 percent, the removal rate of starch reaches 43.11 percent, the degradation rate of protein is 42.27 percent, the removal rate of SCOD reaches 24.58 percent, and the total weight loss rate reaches 60.41 percent. The material added with the expanded FW1 microbial inoculum has a heating rate of 65 ℃/3 days, a crude fat removal rate of 52.77 percent, a starch removal rate of 80.46 percent, a protein degradation rate of 90.27 percent, an SCOD removal rate of 80.73 percent and a total weight loss rate of 90.25 percent. Blank control group: the temperature of the mixed material is raised to 42 ℃, the heating rate is 42 ℃/3 days, the degradation rate of crude fat reaches 20.46 percent, the degradation rate of starch reaches 39.73 percent, the degradation rate of protein reaches 36.28 percent, and the removal rate of SCOD (soluble COD) reaches 20.23 percent; the total weight loss rate reaches 55.48 percent. Although the FW2 treatment effect is obviously better than that of a blank control, the indexes such as temperature rise effect, crude fat removal rate, starch removal rate, SCOD removal rate and the like are all lower than those of FW1 and FW91, the FW91 treatment temperature is highest, the temperature rise is fastest, the starch, protein and SCOD removal rate of FW1 treatment is highest, and the FW91 and FW1 effects are obviously better than those of the blank control and FW2, so FW1 and FW91 strains are reserved, and the composite microbial inoculum is prepared.
The degradation rate of crude fat, starch and protein in a single kitchen waste treatment experiment is calculated as (original index quantity original weight-7 days after experiment each index quantity residual mass)/original index quantity original weight 100%.
The crude fat, starch, protein degradation rates of treatment experiments with continuous kitchen waste addition were calculated as (original respective index amount x original weight +7 daily kitchen index amount x 7 daily addition weight-7 days post experiment respective index amount x residual mass)/(original respective index amount x original weight +7 daily kitchen index amount x 7 daily addition weight) 100%.
TABLE 1 Primary assay screening of three Strain Performance
| Type of microbial inoculum | FW1 | FW2 | FW91 | Blank control |
| Heating rate ℃/3 days | 65 | 52 | 68 | 42 |
| Crude fat degradation Rate% | 52.77 | 40.75 | 58.52 | 20.46 |
| Percent degradation of starch | 73.46 | 43.11 | 74.75 | 39.73 |
| Protein degradation rate% | 70.27 | 42.27 | 70.27 | 36.28 |
| SCOD％ | 80.73 | 24.58 | 79.45 | 20.23 |
| Total weight loss% | 70.25 | 50.41 | 68.45 | 45.48 |
4. Identification of strains
The two strains FW1 and FW91 which are obtained for efficiently degrading the kitchen waste are extracted to obtain DNA of the strain, 16SrDNA fragments are amplified through PCR, DNA sequences are subjected to BLAST comparison on an NCBI database after sequencing, the similarity of the DNA sequence of the FW91 strain and Bacillus subtilis FW91 reaches 99%, the strain is gram-positive bacteria and spherical, and bacterial colonies are in milky opaque dotted distribution (shown in figures 1 and 2). The similarity of the DNA sequence alignment of the FW1 strain and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FW1 reaches 99 percent. The bacteria are gram-positive bacteria, are spherical, and have milky opaque and dotted colonies (shown in figures 2 and 3); the strains FW1 and FW91 have been preserved in China general microbiological culture Collection center (CGMCC) at 12/8 of 2021; bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FW1 with a preservation number of: CGMCC No. 23138; bacillus subtilis FW91 with a preservation number of: CGMCC No. 23139.
Example 2: expanding culture of strain
The method comprises the following steps of (1) expanding and culturing Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1 strains, wherein the steps comprise:
1) adjusting the pH value of the expanding culture medium to 7;
2) after high-pressure steam sterilization, inoculating Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1 in a super clean workbench, placing the workbench at 37 ℃ and 120r/min for shake culture for 48 hours until the culture solution is completely turbid (the turbidity is 0.5 McLeod), wherein the effective bacteria content of the culture solution is more than 10 8 -10 9 cfu/mL。
Wherein, the formula for preparing the culture medium for expanding culture is as follows: 20g of kitchen waste dried powder, 0.05 percent of dipotassium hydrogen phosphate, 0.05 percent of magnesium sulfate, 0.2 percent of 1 percent ferric trichloride solution, 0.05 percent of ammonium sulfate, 0.05 percent of yeast powder, 0.05 percent of sodium chloride and 1000ml of distilled water.
Example 3: composite preparation microbial inoculum and preparation method thereof
Expanding and culturing a strain of bacillus subtilis FW91 and bacillus amyloliquefaciens FW1 according to claim 3 respectively to obtain an expanded strain of bacillus subtilis FW91 and an expanded bacillus amyloliquefaciens FW1, mixing the expanded bacillus subtilis FW91 and the expanded bacillus amyloliquefaciens FW1 according to the weight ratio of 1-2: 1-2, culturing at 37 ℃ and 150-200 rpm for 16-24 hours to obtain the bacillus amyloliquefaciens strain, wherein the culture solution is turbid and cloudy under the illumination of light, and the content of effective bacteria of FW91 and FW1 in the ultraviolet culture solution reaches more than 10 9 cfu/mL。
Example 4: preparing a microbial inoculum FW 91: FW1 is 1: effect of batch run under 1 Condition
Bacillus subtilis FW91, Bacillus amyloliquefaciens FW1 composite bacteria for kitchen waste treatment experiment:
preparing a microbial inoculum FW 91: FW1 is 1: effect of batch run under 1 Condition
Adjusting the carbon-nitrogen ratio to be 25:1, taking 4kg of kitchen waste with the water content of about 80%, fully mixing with 2kg of sawdust (with the water content of about 10%), and placing in a fermentation reactor. Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1 were mixed in a ratio of 1:1, preparing a composite microbial inoculum by compounding, wherein the composite microbial inoculum and materials are mixed according to a weight ratio of 1: 10 concentration proportioning experiment, after the mixture is prepared, tap water is used for adjusting the water content of the fermentation material to be about 65%, and the total weight of the mixture is 7.6 kg. The mixed materials are stirred by an automatic machine at a stirring speed of 15r/min, the mixing of the strains and the materials is strengthened, and in order to ensure the using effect of the microbial inoculum, the temperature, the quality, the fat, the protein, the starch, the moisture content and other index changes of the mixed materials are periodically detected by blowing and supplying oxygen. The fermentation scheme is detailed in FIG. 5.
TABLE 2 biodegradation test parameter Change under a treatment load of 4 kg/batch
From the data in table 2, batch operation under the condition of a processing load of 4kg, complex preparation microbial inoculum FW 91: FW1 ═ 1: under the condition of 1, the weight reduction is 95 percent (the planned saw powder does not participate in degradation reaction), the protein reduction is 79.37 percent, the fat reduction is 67.56 percent, and the starch reduction is 84.12 percent.
Example 5: continuous operation effect under the condition of treating initial load of 4kg
Adjusting according to the carbon-nitrogen ratio of 25:1, taking 4kg of original kitchen waste, and fully mixing with 2kg of sawdust to obtain a mixed material; placing the mixed material in a fermentation reactor, and mixing Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1 in a ratio of 1:1 preparing a composite microbial inoculum in a composite way, wherein the composite microbial inoculum and a mixed material are mixed in a proportion of 1: 10 concentration proportioning experiment, and adjusting the water content of the mixed material to about 65 percent, wherein the total weight is 7.6 kg. The mixed materials are stirred by an automatic machine at a stirring speed of 15r/min, the mixing of strains and the materials is strengthened, meanwhile, the air is blown to supply oxygen, 400g of kitchen waste is fed every day, and the temperature, the quality, the fat, the protein, the starch, the water content and other index changes of the mixed materials are detected regularly.
TABLE 3 treatment of the biodegradation Experimental parameter Change at 4kg initial load/batch
400g of kitchen waste is fed every day, during a 7-day test period, the amount of the subsequently added kitchen waste is 2.4kg, and is increased by 60% compared with the previous case, the data in the table 2 shows that the continuous operation is carried out under the condition of treating the initial load of 4kg, the mass reduction is 68.75%, and under the condition of newly adding the kitchen waste every day, the content of protein, fat and starch on the 7 th day is close to that of the previous case, which indicates that the added kitchen waste is completely degraded and not accumulated, and the reduction of each index is increased compared with that of the previous case.
Example 6: control experiment
The experimental period is 30 days, and in the group I experiment, the ratio of Bacillus subtilis FW91 to Bacillus amyloliquefaciens FW1 is 1:1, preparing a microbial inoculum by compounding, namely initially adding 4kg of kitchen waste, fully mixing with 1kg of sawdust, adjusting the water content to about 55-65%, and subsequently adding 5kg of kitchen waste every 5 days; in group II experiments, the ratio of Bacillus subtilis FW91 to Bacillus amyloliquefaciens FW1 was 1: 2, preparing a microbial inoculum in a compounding way; no microbial inoculum is added in the group III experiments. Three sets of experiments were carried out using the same process conditions as in example 5, and the change in cumulative amount after 30 days is shown in FIG. 6. 30 kilograms of kitchen waste is added in an accumulated mode, and the weight reduction effect of the kitchen waste in the experiment of the group I is obviously superior to that of the experiment of the group II and the experiment of the group III without adding a microbial inoculum.
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 to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A bacterial strain for biological drying of kitchen waste is characterized in that the bacterial strain is Bacillus subtilis or Bacillus amyloliquefaciens; the Bacillus subtilis (Bacillus subtilis) is named as FW91 and has a preservation number of: CGMCC No. 23139; the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is named as FW1 and has the preservation number as follows: CGMCC No. 23138.
2. The method for screening the strains for biological drying of the kitchen waste, which is based on the claim 1, is characterized by comprising the following steps:
step 1: sampling and domesticating culture of kitchen waste: taking the kitchen waste and the sawdust, adjusting the weight ratio of carbon to nitrogen (15-35) to 1, fully mixing, and adjusting the water content to 55-65% to obtain a mixed material; culturing and domesticating the mixed material by aerobic fermentation for 5-9 days, and heating the mixed material to 65-70 deg.C to obtain cultured and domesticated mixed material;
step 2: separating and screening strains: mixing the cultured and domesticated mixed material with sterile water according to the weight ratio of 1 (4-5), and repeatedly and violently shaking to form mixed bacteria liquid; diluting the mixed bacterial liquid to 10% by adopting a multiple dilution method -1 To 10 -7 Gradient bacterial suspension, namely taking the bacterial suspension with the same volume and each dilution degree to carry out scraper coating on a solid culture medium taking kitchen waste as a carbon source, observing the colony morphology in a constant temperature incubator at 40-50 ℃, selecting a well-grown colony in the solid culture medium by comparing the colony morphology, repeatedly carrying out streak separation and purification until the colony morphology characteristics obtained each time are consistent, and obtaining three strains which are named as FW1, FW2 and FW91 respectively;
and step 3: three strains were rescreened by performance assay: respectively carrying out amplification culture on FW1, FW2 and FW91 strains to obtain an amplified FW1 microbial inoculum, an amplified FW2 microbial inoculum and an amplified FW91 microbial inoculum, respectively mixing the amplified FW1 microbial inoculum, the amplified FW1 microbial inoculum and the amplified FW91 microbial inoculum with mixed materials, and carrying out aerobic fermentation; by comparing the crude fat degradation rate, starch degradation rate, protein degradation rate, SCOD removal rate and total weight loss rate of the expanded FW1 microbial inoculum, FW2 microbial inoculum and FW91 microbial inoculum, FW1 and FW91 bacterial strains with remarkable effects, namely bacterial strains for biological drying of kitchen waste, are obtained.
3. The screening method of the bacterial strain for the biological drying of the kitchen waste according to claim 2, wherein the bacterial strain propagation method in the step 3 is as follows: 1) adjusting the pH of the culture medium to 6.5-7.5, and sterilizing with high pressure steam;
2) inoculating strains in a clean bench, performing shake culture at 33-40 deg.C and 120r/min for 36-60 hr until the culture solution of the propagation culture medium is completely turbid, and the turbidity is 0.4-0.6 McLeod, wherein the effective bacteria content of the culture solution of the propagation culture medium reaches 10 7 -10 9 cfu/mL, completing the strain expansion culture.
4. The method for screening the strains for the biological drying of the kitchen waste according to claim 3, wherein the solid culture medium is: 20g of kitchen waste dried powder, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 0.2% of 1% ferric trichloride solution, 0.05% of ammonium sulfate, 0.05% of yeast powder, 0.05% of sodium chloride, 1.5% of agar and 1000ml of distilled water;
the culture medium for expanding culture is as follows: 20g of kitchen waste dried powder, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 0.2% of 1% ferric trichloride solution, 0.05% of ammonium sulfate, 0.05% of yeast powder, 0.05% of sodium chloride and 1000ml of distilled water.
5. A composite microbial inoculum of strains for biological drying of kitchen waste, which is characterized in that the composite microbial inoculum comprises bacillus subtilis FW91 and bacillus amyloliquefaciens FW1 in claim 1, and the effective bacteria content ratio of the bacillus subtilis FW91 to the bacillus amyloliquefaciens FW1 is (1-2) to (1-2).
6. The compound microbial inoculum of the strains for the biological drying of the kitchen waste as claimed in claim 5, wherein the effective bacteria content ratio of the bacillus subtilis FW91 to the bacillus amyloliquefaciens FW1 is 1: 1.
7. The complex microbial inoculant for the biological drying bacterial strains of kitchen waste, as claimed in claim 5, is characterized by consisting of the following components in parts by weight: bacillus subtilis FW 910.5-2%, bacillus amyloliquefaciens FW 10.5-2%, dipotassium hydrogen phosphate 0.04-0.06%, magnesium sulfate 0.04-0.07%, 1% ferric chloride solution 0.15-0.3%, ammonium sulfate 0.04-0.06%, yeast powder 0.04-0.06%, sodium chloride 0.03-0.06% and the balance of water.
8. The compound microbial inoculum of the strains for the biological drying of the kitchen waste according to claim 5, which is characterized by comprising the following components in parts by weight: bacillus subtilis FW 911-1.5%, bacillus amyloliquefaciens FW 11-1.5%, dipotassium hydrogen phosphate 0.05-0.06%, magnesium sulfate 0.05-0.06%, 1% ferric chloride solution 0.20-0.25%, ammonium sulfate 0.04-0.05%, yeast powder 0.04-0.05%, sodium chloride 0.0-0.05%, and the balance of water.
9. A preparation method of a composite microbial inoculum of a strain for biological drying of kitchen waste is characterized in that the strain Bacillus subtilis FW91 and Bacillus amyloliquefaciens FW1 are respectively subjected to amplification culture according to claim 3 to obtain an amplified strain Bacillus subtilis FW91 and an amplified Bacillus amyloliquefaciens FW1, and then the amplified strain Bacillus subtilis FW91 and the amplified Bacillus amyloliquefaciens FW1 are cultured for 16-24 hours at 30 +/-5 ℃ and 150-200 rpm according to the weight ratio of (1-2) to obtain the composite microbial inoculum.
10. The application of the complex microbial inoculant according to any one of claims 5 to 8, wherein the complex microbial inoculant is applied to kitchen waste treatment.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116022911B (en) * | 2022-12-28 | 2023-08-22 | 物产中大(桐庐)水处理有限公司 | Method for improving biological dephosphorization and denitrification efficiency of low-carbon source sewage |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396973A (en) * | 2013-08-22 | 2013-11-20 | 厦门大学 | Method for producing mycoprotein feed through microbial fermentation of kitchen wastes |
| KR20130130413A (en) * | 2012-05-22 | 2013-12-02 | 한국과학기술원 | Microbial community analyses composition for food-waste vanishment and application thereof |
| KR20150107004A (en) * | 2014-03-12 | 2015-09-23 | 주식회사 템셀코리아 | Microorganism for decomposing a food waste, a composition comprising the same and the eliminating method of a food waste using the same |
| WO2019045493A1 (en) * | 2017-08-31 | 2019-03-07 | Cj Cheiljedang Corporation | Novel bacillus amyloliquefaciens strain and method for preparing fermented soy product using the same |
| KR20190043989A (en) * | 2017-10-19 | 2019-04-29 | 재단법인 전라북도생물산업진흥원 | Mixed strain for decomposing food waste and Decomposition method for food waste using same |
| CN110894477A (en) * | 2019-09-18 | 2020-03-20 | 浙江工业大学 | Compound microbial inoculum for degrading kitchen waste, application and kitchen waste degradation method |
| CN111057656A (en) * | 2019-12-02 | 2020-04-24 | 轻工业环境保护研究所 | Yeast for efficiently degrading waste liquid in ice cream production and application thereof |
| CN112011491A (en) * | 2020-09-17 | 2020-12-01 | 西南大学 | Kitchen waste degrading agent |
| CN112174337A (en) * | 2020-09-28 | 2021-01-05 | 西南大学 | Application of kitchen waste fermentation liquor in sewage treatment |
| CN112708579A (en) * | 2021-01-15 | 2021-04-27 | 杭州鹏创环保科技有限公司 | Composite strain and application thereof in degradation of kitchen waste |
| AU2021103470A4 (en) * | 2020-07-09 | 2021-08-05 | Hebei Haoyuan Environmental Engineering Co., Ltd. | Bacillus subtilis bs40-4 strain and method for composting organic wastes by using the same |
| WO2021254117A1 (en) * | 2020-06-16 | 2021-12-23 | 金华康扬环境科技有限公司 | Bacillus velezensis ky01 and application thereof in degradation of kitchen garbage |
-
2022
- 2022-06-08 CN CN202210644888.7A patent/CN115058357A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130130413A (en) * | 2012-05-22 | 2013-12-02 | 한국과학기술원 | Microbial community analyses composition for food-waste vanishment and application thereof |
| CN103396973A (en) * | 2013-08-22 | 2013-11-20 | 厦门大学 | Method for producing mycoprotein feed through microbial fermentation of kitchen wastes |
| KR20150107004A (en) * | 2014-03-12 | 2015-09-23 | 주식회사 템셀코리아 | Microorganism for decomposing a food waste, a composition comprising the same and the eliminating method of a food waste using the same |
| WO2019045493A1 (en) * | 2017-08-31 | 2019-03-07 | Cj Cheiljedang Corporation | Novel bacillus amyloliquefaciens strain and method for preparing fermented soy product using the same |
| KR20190043989A (en) * | 2017-10-19 | 2019-04-29 | 재단법인 전라북도생물산업진흥원 | Mixed strain for decomposing food waste and Decomposition method for food waste using same |
| CN110894477A (en) * | 2019-09-18 | 2020-03-20 | 浙江工业大学 | Compound microbial inoculum for degrading kitchen waste, application and kitchen waste degradation method |
| CN111057656A (en) * | 2019-12-02 | 2020-04-24 | 轻工业环境保护研究所 | Yeast for efficiently degrading waste liquid in ice cream production and application thereof |
| WO2021254117A1 (en) * | 2020-06-16 | 2021-12-23 | 金华康扬环境科技有限公司 | Bacillus velezensis ky01 and application thereof in degradation of kitchen garbage |
| AU2021103470A4 (en) * | 2020-07-09 | 2021-08-05 | Hebei Haoyuan Environmental Engineering Co., Ltd. | Bacillus subtilis bs40-4 strain and method for composting organic wastes by using the same |
| CN112011491A (en) * | 2020-09-17 | 2020-12-01 | 西南大学 | Kitchen waste degrading agent |
| CN112174337A (en) * | 2020-09-28 | 2021-01-05 | 西南大学 | Application of kitchen waste fermentation liquor in sewage treatment |
| CN112708579A (en) * | 2021-01-15 | 2021-04-27 | 杭州鹏创环保科技有限公司 | Composite strain and application thereof in degradation of kitchen waste |
Non-Patent Citations (3)
| Title |
|---|
| LAIA BLAVI等: "Effects of Bacillus amyloliquefaciens and Bacillus subtilis on ileal digestibility of AA and total tract digestibility of CP and gross energy in diets fed to growing pigs", J ANIM SCI, vol. 92, no. 2, pages 727 - 734 * |
| 吴昊;张赣道;: "生活垃圾处理机生物降解生活垃圾的微生物研究", 安徽农业科学, no. 26, pages 269 - 270 * |
| 林杨;刘淼;林锋;张宿义;霍丹群;陈飞;杨艳;李德林;李嘉伟;: "微生物絮凝剂研究进展及其在食品工业中的应用", 中国酿造, no. 01, pages 1 - 12 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116022911B (en) * | 2022-12-28 | 2023-08-22 | 物产中大(桐庐)水处理有限公司 | Method for improving biological dephosphorization and denitrification efficiency of low-carbon source sewage |
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