CN112625959A - Thermophilic composite microbial inoculum for aerobic fermentation of organic wastes - Google Patents
Thermophilic composite microbial inoculum for aerobic fermentation of organic wastes Download PDFInfo
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Abstract
The invention discloses a thermophilic composite microbial inoculum for aerobic fermentation of organic wastes, which comprises 15-20 vol% of streptococcus thermophilus, 20-25 vol% of bacillus subtilis, 20 vol% of bacillus licheniformis, 20 vol% of bacillus denitrificans and 20 vol% of brevibacillus borstelensis, wherein the bacillus licheniformis and the brevibacillus borstelensis are preserved. According to the thermophilic composite microbial inoculum, all thermophilic bacteria grow rapidly within 24 hours, and the growth is fast, so that strong growth activity can be kept at a high-temperature stage in aerobic fermentation, and the rapid fermentation of organic wastes is realized.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a thermophilic composite microbial inoculum for aerobic fermentation of organic wastes.
Background
The kitchen waste in China has huge output, and the quantity of the kitchen waste in clearance per year exceeds 6000 million t according to the estimation that the kitchen waste accounts for 30% -40% of the urban domestic waste. In a part of super-huge cities, the proportion of kitchen garbage is as high as 60 percent, and the highest proportion can be more than 80 percent. The traditional kitchen waste recycling technology is used for preparing various feeds, but the technology is gradually limited or prohibited by governments of various countries due to various potential safety hazards, and a new kitchen waste recycling mode is gradually concerned. The aerobic composting technology can realize the harmlessness and reduction of kitchen garbage and can produce a safe, stable and nutrient-rich soil conditioner. The realization of the food waste composting by the high-temperature aerobic digestion technology is one of the recent research hotspots. Under the high-temperature condition (45-55 ℃), the technology can accelerate the metabolism of microorganisms, improve the degradation speed of organic substances in the kitchen waste, rapidly and efficiently degrade the kitchen waste into organic fertilizers, and can kill most pathogenic bacteria and parasites at higher temperature to realize the stabilization and recycling of the waste.
In the aerobic composting treatment process of organic solid wastes, the reasonable addition of the efficient microbial agent is beneficial to enhancing the function of a microbial ecosystem, accelerating the decomposition of organic substances, promoting the decomposition of composting materials and improving the composting efficiency. The thermophilic bacteria have higher microbial metabolic activity and organic matter degradation rate than the mesophilic bacteria, and have wide application prospect and scientific value in the field of solid waste treatment. Thermophilic microorganisms can secrete protease, amylase and other extracellular enzymes with excellent thermal stability, and show good cell lytic enzyme activity in macromolecular degradation of kitchen waste.
At present, microbial agents applied to compost mainly comprise single microbial agents and complex microbial agents, wherein the single microbial agents mainly comprise Bacillus (Bacillus) and Actinomycetes (Actinomycetes) with strong heat resistance and the like. But the application effect of the single strain in the compost is limited due to low environmental adaptability of the single strain. The composite microbial inoculum generated based on the mutualistic symbiotic effect among strains has strong capability of adapting to high-temperature composting environment, and the application effect is obviously better than that of composting treatment inoculated with a single microbial inoculum. The addition of the compound microbial agent can not only shorten the composting time, but also make the materials become a micro-ecological environment suitable for the growth of microorganisms. At present, few microbial inoculums are applied to high-temperature aerobic fermentation in organic waste treatment.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a thermophilic composite microbial inoculum for aerobic fermentation of organic wastes, which can accelerate degradation of organic matters, promote fermentation and decomposition of piles and shorten composting time; increasing the temperature of the stack body and killing the ova and germs contained in the excrement; and (3) degrading substances such as antibiotics and the like remained in the stacked materials.
The purpose of the invention is realized by the following technical scheme:
a thermophilic composite microbial inoculum for aerobic fermentation of organic wastes comprises:
15-20 vol% of streptococcus thermophilus
20-25 vol% of bacillus subtilis
Bacillus licheniformis 20 vol%
Geobacillus thermodenitrificans 20 vol%
Brevibacillus borstelensis 20 vol%;
wherein, the Bacillus licheniformis (Bacillus licheniformis) VH1-1 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation address: no. 3 of No. 1 Hospital of Beijing, Chaoyang, on the North road, the preservation date is 26 days 11 months after 2020, and the preservation number is CGMCC No. 21231;
the Brevibacillus borstelensis (Brevibacillus borstelensis) VH1-3 is preserved in China general microbiological culture Collection center, and the preservation address is as follows: no. 3 of Beijing, Chaoyang district, Beichen Lu No. 1, the preservation date is 26 days 11 months 2020, and the preservation number is CGMCC No. 21232.
Further, the volume ratio of the streptococcus thermophilus, the bacillus subtilis, the bacillus licheniformis, the bacillus denitrificans and the brevibacillus borstelensis is 15 vol%, 25 vol%, 20 vol% and 20 vol%.
Further, the volume ratio of the streptococcus thermophilus, the bacillus subtilis, the bacillus licheniformis, the bacillus denitrificans and the brevibacillus borstelensis is 20 vol%, 20 vol% and 20 vol%.
The invention has the following beneficial effects:
1. according to the composite microbial inoculum, all high-temperature bacteria grow rapidly within 24 hours, and the growth activity can be kept stronger in a high-temperature stage in aerobic fermentation by utilizing the rapid growth of the high-temperature bacteria, so that the rapid fermentation of organic wastes is realized.
2. The invention can be applied to kitchen waste treatment equipment with heating, aerating and stirring functions, can grow and play a role at 50-60 ℃, and leads the kitchen waste to complete aerobic fermentation within 3 days and reach a completely decomposed state (the germination index is more than 80%).
Drawings
FIG. 1 is a morphological diagram of two thermophilic bacteria, wherein FIG. 1(a) is a morphological diagram of L6-5-36, and FIG. 1(b) is a morphological diagram of FF 2-4-2;
FIG. 2 is a histogram of the decrement of kitchen waste with time obtained from three experiments;
FIG. 3 is a histogram of changes in water content and power consumption of the kitchen waste obtained from three experiments;
fig. 4 is a bar graph of the change of the pH value and the germination percentage of the kitchen waste obtained by three groups of experiments.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments, and the objects and effects of the present invention will become more apparent, it being understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
The high-temperature complex microbial inoculant for aerobic fermentation of organic wastes comprises streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus denitrificans and brevibacillus borstelensis, wherein the volume ratio of the five bacteria is 15-20%: 20-25%: 20%: 20%: 20 percent;
the Bacillus licheniformis (Bacillus licheniformis) VH1-1 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 11-26 months in 2020, and the preservation number is CGMCC No. 21231;
the Brevibacillus borstelensis (Brevibacillus borstelensis) VH1-3 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 11-26 months in 2020, and the preservation number is CGMCC No. 21232.
The other three bacteria are purchased from China general microbiological culture Collection center. The culture media used in the examples of the present invention were as follows:
(1) beef extract peptone medium: 5g of beef extract, 10g of peptone, 5g of NaCl, 1000mL of H2O 1000, 20g of agar, 7.0-7.2 of pH and 20min of sterilization at 121 ℃.
(2) LB solid medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar, 1000mL of deionized water and pH 7.4.
(3) LB liquid medium: LB broth 20g, deionized water 1000 mL.
The process of separating, screening and purifying strains in the embodiment of the invention is as follows:
1. domestication of strains
Mixing the kitchen waste and the high organic soil according to the volume ratio of 1:1, and then putting the mixture into an oven at 50 ℃/60 ℃ for culture, namely enrichment culture. The primary culture mainly comprises the sample of the windrow compost in the high-temperature period. Diluting the enrichment culture sample and the primary culture sample, coating the dilution culture sample on a beef extract peptone culture medium, and freezing and storing glycerol at-80 ℃ after streaking and picking single bacteria.
(1) Separation, screening and purification of Bacillus licheniformis VH1-1
And (3) performing gradient dilution on the bacterial suspension of the strip pile compost high-temperature-stage sample, performing streak culture on a beef extract peptone solid culture medium, performing separation and purification for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as VH 1-1.
Bacterial colonies of the strain VH1-1 are in a shape of a sharp circle tooth, are milk white, have moist and glossy surfaces and are abundant in mucus, and are large in size and 4-5 mm in diameter. The optimal culture temperature is 60 ℃, the bacillus subtilis is gram-positive rod-shaped bacteria, can generate endogenetic spores, has strong heat resistance and stress resistance, and is ubiquitous on the surfaces of soil and plants. Is a common endophyte in plants, is nontoxic and harmless to people and livestock, and does not pollute the environment. High growth speed and simple nutritive requirement. As shown in fig. 1.
(2) Separation, screening and purification of Brevibacillus borstelensis VH1-3
And (3) carrying out gradient dilution on the bacterial suspension of the enrichment culture sample, carrying out streak culture on a beef extract peptone solid culture medium, carrying out separation and purification for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as VH 1-3.
Bacterial colony of the strain VH1-3 is round, milky white, smooth in surface, opaque and slightly raised. The optimum culture temperature is 50 ℃, the physiological and biochemical characteristics are that gelatin liquefaction, nitrate reduction citrate utilization and glucose are positive, and oxidase, starch hydrolysis and V-P reaction are negative. As shown in fig. 1.
2. Strain isolation and identification
The 16S rRNA gene of the strain is amplified by PCR by taking the genome DNA of the strain obtained by separation and purification of 2 culture modes as a template and 1492R and 27F as primers, the amplified product is sequenced, the obtained sequence is classified by online ezTaxon (the 16S rRNA gene sequence similarity is more than 97 percent and is primarily determined to be the same species), and the similarity between the 16S rRNA gene sequence of the obtained 2 strains of bacteria and the model species is 98-100 percent. The results are shown in Table 1.
TABLE 1 analysis of the similarity of the 16S rRNA genes of thermophilic hyperthermophilic strains
| Strain numbering | Degree of similarity | Identification results |
| L6-5-36 | 99.10％ | Bacillus_licheniformis(CP000002) |
| FF2-4-2 | 100.00％ | Brevibacillus_borstelensis(AB112721) |
3. Expanded culture of strains
Selecting a loop of the streptococcus thermophilus strain stored on the test tube slant by using an inoculating loop, and carrying out amplification culture in an LB liquid culture medium at the culture temperature of 60 ℃, the oscillation speed of 180rpm/min and the culture time of 18 h.
Selecting a loop of the bacillus subtilis strain preserved on the test tube slant by using an inoculating loop, and carrying out amplification culture in an LB liquid culture medium at the culture temperature of 50 ℃, the oscillation speed of 180rpm/min and the culture time of 18 h.
Selecting a loop of the bacillus licheniformis strain preserved by freezing glycerin at the temperature of minus 80 ℃ by using an inoculating loop, and carrying out amplification culture in an LB liquid culture medium at the culture temperature of 60 ℃, the oscillation speed of 180rpm/min and the culture time of 18 h.
A loop of the strain of the Geobacillus thermodenitrificans preserved on the inclined plane of the test tube is picked by using an inoculating loop, and amplification culture is carried out in an LB liquid culture medium at the culture temperature of 60 ℃, the oscillation speed of 180rpm/min and the culture time of 18 h.
Selecting a loop of Brevibacillus borstelensis strain frozen and preserved at minus 80 ℃ by using an inoculating loop, and carrying out amplification culture in an LB liquid culture medium at the culture temperature of 50 ℃, the oscillation speed of 180rpm/min and the culture time of 18 h.
4. Detecting the colony count of the mixed bacterial liquid
Selecting the bacterial liquid of five bacteria for amplification culture10mL, as a primary seed culture medium. Adjusting the OD600 of the primary seed culture solution to the same level, transferring 5mL of the seed culture solution to 150mL of liquid culture medium, and performing shake culture for 20h to obtain a secondary seed culture solution. Taking 1ml of bacterial liquid, measuring OD600, diluting bacterial liquid to 10-1、10-2、10-3、10-4、10-5、10-6The number of different colonies was determined by dot-blot method using a pipette with a gradient of 10 from each dilution-3、10-4、10-5、10-6The centrifugal tube absorbs 10 mul of diluent to be uniformly spotted in corresponding areas in the plate, 10 mul of bacterial liquid in each area is spotted at 5-6 points, the plate is naturally aired and then is placed in a constant temperature incubator to be cultivated at the optimum cultivation temperature of each bacterium, when bacterial colonies grow to the appropriate size, counting is carried out, and each treatment is carried out in three-time mode. The results of the experiments are shown in the following table.
TABLE 2 statistical table of colony counts of five bacteria
| Strain name | Strain numbering | OD600 | Number of colonies |
| Streptococcus thermophilus | 1 | 1.086 | 8*10^8 |
| Bacillus subtilis | 2 | 1.071 | 6*10^8 |
| Bacillus licheniformis | L6-5-36(VH1-1) | 1.533 | 6*10^8 |
| Thermophilic denitrifying bacillus | 3 | 1.056 | 6*10^8 |
| Brevibacillus borstelensis | FF2-4-2(VH1-3) | 1.154 | 4*108 |
As can be seen from Table 2, the colony counts of the five bacteria were each greater than 108And meets the requirement of preparing the complex microbial inoculum.
Example 1
(2) Uniformly mixing five bacteria solutions for enlarged culture, wherein the volume percentage of each bacteria solution is as follows: 20% of streptococcus thermophilus, 20% of bacillus subtilis, 20% of bacillus licheniformis, 20% of bacillus denitrificans and 20% of brevibacillus borstelensis, thus obtaining the composite microbial inoculum.
Example 2
(1) Uniformly mixing five bacteria solutions for enlarged culture, wherein the volume percentage of each bacteria solution is as follows: 15% of streptococcus thermophilus, 25% of bacillus subtilis, 20% of bacillus licheniformis, 20% of bacillus denitrificans and 20% of brevibacillus borstelensis, thus obtaining the composite microbial inoculum.
In order to demonstrate the effect of the complex microbial inoculum of the present invention, a comparative experiment was conducted as follows.
Comparative experiment 1
The complex microbial inoculum prepared in example 1 and a commercially available microbial inoculum S are respectively inoculated into a 500mL conical flask filled with 100g of kitchen waste, and treated at 50 ℃ and an oscillation speed of 180rpm/min for 7 days, 16:00 continuously sampling and measuring the decrement of the kitchen waste. The decrement effect is recorded in a weighing manner. Meanwhile, a control group without adding the microbial inoculum is arranged. The weight reduction results obtained are shown in fig. 2.
CK: no addition of microbial inoculum
VH1 (M): complex microbial inoculum obtained in example 1
S: adding commercial Sukohamia agent.
As shown in FIG. 1, the weight reduction effect is not obvious on day 1 due to too short time, and the weight reduction of 3 treatments is 1-4 per mill. With the change of time, on day 7, the amount of the kitchen waste in the conical flask added with the complex microbial inoculum of example 1 is 5% higher than that of the control group. It can be seen that the reduction effect by adding the complex microbial inoculum of example 1 is the best.
Comparative experiment 2
The compound microbial inoculum obtained in the example 2 and a commercially available microbial inoculum S are respectively inoculated into 50L kitchen waste treatment equipment filled with 30Kg of kitchen waste, the addition amount is 5 per mill, and 10% of sawdust is added for adjusting the water content of the materials. Equipment parameter setting indexes: after materials are added into the reactor, the set parameters are that the temperature of a heating plate is 50 ℃, the temperature of a biochemical cabin is 60 ℃, and the stirring parameters are as follows: rotating forward for 20min, rotating backward for 20min, and stopping for 20 min; after incubation for 2h, the operation program is adjusted to be that the temperature of a heating plate is 100 ℃, the temperature of a biochemical cabin is 105 ℃, and the stirring parameters are as follows: rotating forward for 20min, rotating backward for 20min, and stopping for 20 min; and setting the aeration and air draft interval at 50-55 ℃, continuously operating for 72 hours, continuously sampling at a rate of 16:00 per day, and measuring the water content, pH, GI and power consumption of the kitchen waste as shown in figures 3-4.
As can be seen from FIG. 3, the initial water content of each treated material is about 79%, and after 3 days of biological drying treatment, the water content is 31.42%, 37.64% and 32.98%, respectively, which indicates that the water content is reduced more rapidly after the microbial inoculum is added. The reduction is most obvious when VH1(M) microbial inoculum is added. Among the three treatments, the power consumption of the complex microbial inoculum obtained in example 2 was the lowest. The later period may be due to the fact that the power consumption is low due to factors such as reduction of water content of materials, increase of water transmission efficiency, increase of capability of maintaining temperature of decomposition and heat release of microorganisms and the like. It follows that the higher the water content, the higher the energy consumption required.
As can be seen from FIG. 4, the kitchen waste added with VH1(M) has a fast pH increase rate, but the pH change of each treatment is basically 5-5.4, probably because acidic substances are decomposed by microorganisms, the pH slightly increases, probably because the biological drying rate is too high, the water content decreases too fast, the heap environment is not suitable for the growth of microorganisms, and the pH does not increase to 7.0 or above. In the whole biological drying process, VH1(M) microbial inoculum is added to treat the germination rate to reach more than 80, so that the requirement of decomposition is met.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and although the invention has been described in detail with reference to the foregoing examples, it will be apparent to those skilled in the art that various changes in the form and details of the embodiments may be made and equivalents may be substituted for elements thereof. All modifications, equivalents and the like which come within the spirit and principle of the invention are intended to be included within the scope of the invention.
Claims (3)
1. A thermophilic composite microbial inoculum for aerobic fermentation of organic wastes is characterized by comprising the following components in parts by weight:
15-20 vol% of streptococcus thermophilus
20-25 vol% of bacillus subtilis
Bacillus licheniformis 20 vol%
Geobacillus thermodenitrificans 20 vol%
Brevibacillus borstelensis 20 vol%.
Wherein the Bacillus licheniformis (Bacillus licheniformis) VH1-1 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 26 months at 11 months in 2020 and the preservation number of CGMCC No. 21231;
the Brevibacillus borstelensis (Brevibacillus borstelensis) VH1-3 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 11-26 months in 2020, and the preservation number is CGMCC No. 21232.
2. The thermophilic complex microbial inoculant for aerobic fermentation of organic waste as claimed in claim 1, wherein the volume ratio of streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus denitrificans and brevibacillus borstelensis is 15 vol%, 25 vol%, 20 vol% and 20 vol%.
3. The thermophilic composite microbial inoculant for aerobic fermentation of organic waste as claimed in claim 1, wherein the volume ratio of streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus denitrificans and brevibacillus borstelensis is 20 vol%, 20 vol% and 20 vol%.
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| CN112522164A (en) * | 2020-12-25 | 2021-03-19 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Forest fruit branch decomposing inoculant and preparation method and application thereof |
| CN113416097A (en) * | 2021-07-22 | 2021-09-21 | 东北大学 | Method for efficiently removing antibiotics and resistance genes and stabilizing heavy metals in livestock and poultry excrement based on ultrahigh-temperature aerobic fermentation technology |
| CN114317323A (en) * | 2021-11-25 | 2022-04-12 | 中农新科(苏州)有机循环研究院有限公司 | Microbial agent for rapidly degrading organic matters in kitchen waste |
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