CN112625959B - Thermophilic composite microbial inoculant for aerobic fermentation of organic waste - Google Patents
Thermophilic composite microbial inoculant for aerobic fermentation of organic waste Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F9/00—Fertilisers from household or town refuse
- C05F9/04—Biological compost
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a thermophilic composite microbial inoculum for aerobic fermentation of organic waste, which comprises 15-20 vol% of streptococcus thermophilus, 20-25 vol% of bacillus subtilis, 20vol% of bacillus licheniformis, 20vol% of bacillus thermodenitrificans and 20vol% of Brevibacillus borstellensis, wherein both the bacillus licheniformis and the Brevibacillus borstellensis are preserved. According to the thermophilic composite microbial inoculum, all the thermophilic bacteria grow rapidly within 24 hours, and the growth is rapid, so that the high growth activity can be kept in 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 inoculant for aerobic fermentation of organic wastes.
Background
The kitchen waste yield in China is huge, the kitchen waste accounts for 30% -40% of the urban household waste, and estimated kitchen waste cleaning and transporting capacity exceeds 6,000 ten thousand t each year. In some extra large cities, the proportion of kitchen waste is up to 60%, and the highest proportion can be up to more than 80%. The traditional kitchen waste recycling technology is used for preparing various feeds, but the technology is gradually limited or forbidden by various governments due to various potential safety hazards, and a new kitchen waste recycling mode is adopted to pay attention. The aerobic composting technology can not only realize the harmless and reduction of kitchen waste, but also produce safe, stable and nutrient-rich soil conditioner. The realization of kitchen waste fertilizer by a high-temperature aerobic digestion technology is one of the recent research hot spots. The technology can quickly and efficiently degrade the kitchen waste into the organic fertilizer by accelerating the metabolism of microorganisms and improving the degradation speed of organic substances in the kitchen waste under the high temperature condition (45-55 ℃), and meanwhile, the technology can kill most pathogenic bacteria and parasites through higher temperature to realize the stabilization and recycling of the waste.
In the aerobic composting treatment process of the organic solid waste, the reasonable addition of the high-efficiency microbial inoculant is beneficial to enhancing the functions of a microbial ecological system, accelerating the decomposition of organic substances, promoting the decomposition of composting materials and improving the composting efficiency. Compared with normal temperature bacteria, the high temperature bacteria have higher microorganism metabolism activity and organic matter degradation rate, and have wide application prospect and scientific value in the field of solid waste treatment. Thermophilic microorganisms can secrete extracellular enzymes such as protease, amylase and the like with excellent thermal stability, and the extracellular enzymes show good cytolytic enzyme activity in the degradation of kitchen waste macromolecules.
At present, the microbial agents applied to composting mainly comprise single microbial agents and composite microbial agents, wherein the single microbial agents mainly comprise Bacillus (Bacillus) with relatively high heat resistance, actinomycetes (Actinomycetes) and the like. But the application effect of the single strain in composting is limited due to the low environmental adaptability of the single strain. The composite microbial inoculum generated based on the reciprocal symbiotic effect among the strains has strong capability of adapting to high-temperature composting environment and obviously better application effect than composting treatment of inoculating a single microbial inoculum. The addition of the composite microbial agent not only shortens the composting time, but also enables the materials to be a micro-ecological environment suitable for microbial growth. At present, the microbial inoculum applied to high-temperature aerobic fermentation for treating organic wastes is less.
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 a heap body and shorten heap preparation time; increasing the temperature of the pile body to kill the oomycete germs contained in the excrement; degrading antibiotics and other substances remained in the pile materials.
The aim of the invention is achieved by the following technical scheme:
a thermophilic composite microbial inoculant for aerobic fermentation of organic waste, the thermophilic composite microbial inoculant comprising:
15 to 20vol% of streptococcus thermophilus
20 to 25vol percent of bacillus subtilis
Bacillus licheniformis 20vol%
Bacillus thermodenitrificans 20vol%
20vol% of Brevibacillus bordetention;
wherein, the bacillus licheniformis (Bacillus licheniformis) VH1-1 is preserved in China general microbiological culture Collection center, and the preservation address is: the preservation date is 11 months and 26 days in 2020, and the preservation number is CGMCC No.21231;
the Brevibacillus bordetention (Brevibacillus borstelensis) VH1-3 is preserved in China general microbiological culture Collection center, and has the preservation address: no. 3 of North Chenli No. 1 in the Chaoyang district of Beijing city, the preservation date is 11 months and 26 days in 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 stearothermophilus and the Brevibacillus bordetention is 15vol%, 25vol%, 20vol% and 20vol%.
Further, the volume ratio of the streptococcus thermophilus, the bacillus subtilis, the bacillus licheniformis, the bacillus stearothermophilus and the Brevibacillus bordetention is 20vol%, 20vol% and 20vol%.
The beneficial effects of the invention are as follows:
1. the composite microbial inoculum provided by the invention has the advantages that all high-temperature bacteria grow rapidly within 24 hours, and the growth is rapid, so that the high growth activity can be kept in a high-temperature stage in aerobic fermentation, and the rapid fermentation of organic wastes is realized.
2. The invention is applicable to kitchen waste treatment equipment with heating, aeration and stirring functions, can grow and play a role at 50-60 ℃, ensures that the aerobic fermentation of the kitchen waste is completed within 3 days, and reaches a fully decomposed state (germination index is more than 80%).
Drawings
FIG. 1 is a diagram showing two types of thermophilic bacteria, wherein FIG. 1 (a) is a diagram showing L6-5-36, and FIG. 1 (b) is a diagram showing FF 2-4-2;
FIG. 2 is a bar graph of kitchen waste reduction obtained by three groups of experiments and changing along with time;
FIG. 3 is a bar graph of the water content and power consumption of the kitchen waste obtained by three groups of experiments;
fig. 4 is a bar graph of the change in ph and germination rate of the kitchen waste obtained by three experiments.
Detailed Description
The objects and effects of the present invention will become more apparent from the following detailed description of the preferred embodiments and the accompanying drawings, it being understood that the specific embodiments described herein are merely illustrative of the invention and not limiting thereof.
The high-temperature composite microbial inoculum is used for aerobic fermentation of organic wastes and comprises streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus stearothermophilus and Brevibacillus borstevens, wherein the volume ratio of the five bacteria is 15-20%: 20-25%: 20%:20%:20% of a base;
the bacillus licheniformis (Bacillus licheniformis) VH1-1 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2020, 11 months and 26 days and a preservation number of CGMCC No.21231;
the Brevibacillus bordetensis (Brevibacillus borstelensis) VH1-3 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 11 months and 26 days in 2020 and a preservation number of CGMCC No.21232.
The other three bacteria were purchased from the China general microbiological culture Collection center, which was a Commission on the preservation of microorganisms. The culture medium used in the embodiment of the invention is as follows:
(1) Beef extract peptone medium: beef extract 5g, peptone 10g, naCl 5g, H2O 1000mL, agar 20g, pH 7.0-7.2, and sterilizing at 121deg.C for 20min.
(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 1000mL.
The separation, screening and purification processes of the strain in the embodiment of the invention are specifically as follows:
1. domestication of strains
Mixing kitchen waste and high organic matter soil according to a volume ratio of 1:1, and then placing the mixture into a baking oven at 50 ℃/60 ℃ for culturing, thus obtaining enrichment culture. The primary culture is mainly a sample of the high-temperature period of the crenelated fertilizer. And diluting and coating the enriched culture sample and the primary culture sample on a beef extract peptone culture medium, streaking and picking single colonies, and freezing and preserving glycerol at-80 ℃.
(1) Separation, screening and purification of bacillus licheniformis VH1-1
And (3) carrying out streak culture on the bacterial suspension of the samples in the high-temperature period of the crenelated fertilizer on a beef extract peptone solid culture medium after gradient dilution, separating and purifying for multiple times to obtain single bacterial colony which is named as VH1-1, and preserving.
Bacterial colony of strain VH1-1 is in the shape of circular sharp teeth, milky white, moist and glossy surface and a large amount of mucus, and has large bacterial colony and diameter of 4-5 mm. The optimal culture temperature is 60 ℃, the culture temperature is gram-positive rod-shaped bacteria, endophytic spores can be generated, the heat resistance and stress resistance are strong, and the culture temperature 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. The growth speed is high, and the nutrition requirement is simple. As shown in fig. 1.
(2) Separation, screening and purification of Brevibacillus borstele VH1-3
Culturing bacterial suspension of enriched culture samples on a beef extract peptone solid culture medium in a streaking way after gradient dilution, separating and purifying for multiple times to obtain single colony which is named as VH1-3, and preserving.
Bacterial strain VH1-3 colonies were round, milky white, smooth, opaque, slightly elevated. The optimal culture temperature is 50 ℃, gelatin liquefaction, nitrate reduction citrate utilization, positive glucose, and negative oxidase, starch hydrolysis and V-P reaction are carried out in physiological and biochemical characteristics. As shown in fig. 1.
2. Separating and identifying strain
The genome DNA of the strain obtained by separating and purifying in 2 culture modes is used as a template, 1492R and 27F are used as primers, the 16S rRNA genes of the strain are amplified by PCR, amplified products are sequenced, the obtained sequences are classified by online EzTaxon (the preliminary determination that the similarity of the 16S rRNA gene sequences is more than 97 percent is the same), and the similarity of the 16S rRNA gene sequences of the obtained 2 strains of bacteria and model seeds is 98-100 percent. The results are shown in Table 1.
TABLE 1 16S rRNA Gene similarity analysis of thermophilic hyperthermophilic strains
| Strain numbering | Similarity degree | Identification result |
| L6-5-36 | 99.10％ | Bacillus_licheniformis(CP000002) |
| FF2-4-2 | 100.00％ | Brevibacillus_borstelensis(AB112721) |
3. Expansion culture of strain
The streptococcus thermophilus strain preserved on the inclined surface of the test tube is picked into a loop by an inoculating loop, amplified culture is carried out in LB liquid culture medium, the culture temperature is 60 ℃, the shaking speed is 180rpm/min, and the culture time is 18 hours.
And (3) picking a loop of bacillus subtilis strain stored on the inclined surface of the test tube by using an inoculating loop, and performing amplification culture in an LB liquid culture medium at a culture temperature of 50 ℃ and a shaking speed of 180rpm/min for 18 hours.
The bacillus licheniformis strain which is frozen and preserved by glycerol at the temperature of minus 80 ℃ is picked into a loop by an inoculating loop, amplified culture is carried out in an LB liquid culture medium, the culture temperature is 60 ℃, the shaking speed is 180rpm/min, and the culture time is 18 hours.
And (3) picking a loop of the thermophilic denitrifying bacillus strain stored on the inclined surface of the test tube by using an inoculating loop, and performing amplification culture in an LB liquid culture medium at the culture temperature of 60 ℃ at the shaking speed of 180rpm/min for 18 hours.
The Brevibacillus borsteii strain frozen and preserved by glycerol at the temperature of minus 80 ℃ is picked into a loop by an inoculating loop, amplified culture is carried out in an LB liquid culture medium, the culture temperature is 50 ℃, the shaking speed is 180rpm/min, and the culture time is 18 hours.
4. Detecting the colony count of the mixed bacterial liquid
10mL of the bacterial liquid of the five bacteria which are subjected to the expansion culture is selected as a primary seed culture liquid. After the OD600 of the primary seed culture solution was adjusted to the same level, 5mL of the seed culture solution was removed and cultured in 150mL of a liquid medium for 20 hours as a secondary seed culture solution. Taking 1ml of bacterial liquid, measuring OD600, diluting the bacterial liquid to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 The number of different colonies was determined by spot grafting and the dilution gradient from each was 10 with a pipette -3 、10- 4 、10 -5 、10 -6 Sucking 10 μl of diluent into centrifuge tube of the culture medium, uniformly spotting in corresponding region of the plate, naturally airing the plate, placing the plate upside down in a constant temperature incubator at optimum culture temperature for each strain, counting when colony grows to proper size, and performing each treatmentThree replicates were set. The experimental results are shown in the following table.
TABLE 2 colony count statistics of five bacteria
| Strain name | Strain numbering | OD600 | Colony count |
| 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 borstele | FF2-4-2(VH1-3) | 1.154 | 4*108 |
As can be seen from Table 2, the colony numbers of the five bacteria were greater than 10 8 Meets the requirement of preparing the composite microbial inoculum.
Example 1
(2) Mixing the five bacterial liquids subjected to the expansion culture uniformly, wherein the percentage content of each bacterial liquid is as follows: 20% of streptococcus thermophilus, 20% of bacillus subtilis, 20% of bacillus licheniformis, 20% of bacillus thermodenitrificans and 20% of Brevibacillus borstellensis to obtain the composite microbial inoculum.
Example 2
(1) Mixing the five bacterial liquids subjected to the expansion culture uniformly, wherein the percentage content of each bacterial liquid is as follows: 15% of streptococcus thermophilus, 25% of bacillus subtilis, 20% of bacillus licheniformis, 20% of bacillus thermodenitrificans and 20% of Brevibacillus borstellensis to obtain the composite microbial inoculum.
In order to demonstrate the effect of the composite microbial agent of the present invention, comparative experiments were performed as follows.
Comparative test 1
The composite bacterial agent and the commercial bacterial agent S prepared in example 1 were inoculated into 500mL Erlenmeyer flasks containing 100g of kitchen waste, and treated at 50℃and 180rpm/min for 7 days with shaking speed of 16:00 continuous sampling to measure the kitchen waste decrement. The decrement effect is recorded in a weighing manner. Meanwhile, a control group without the addition of the microbial inoculum is set. The weight loss results obtained are shown in FIG. 2.
CK: no microbial agent is added
VH1 (M): the composite microbial inoculum obtained in example 1
S: commercial Su Kehan microbial inoculum was added.
As shown in FIG. 1, the reduction effect is not obvious on day 1 due to the too short time, and the reduction of 3 treatments is 1-4 per mill. On day 7, the reduction of kitchen waste in the conical flask to which the composite microbial inoculum of example 1 was added was 5% higher than that of the control group. It can be seen that the reduction effect of the composite microbial agent of example 1 was optimal.
Comparative test 2
The composite bacterial agent and the commercial bacterial agent S obtained in the example 2 are respectively inoculated into 50L kitchen waste treatment equipment filled with 30Kg kitchen waste, the addition amounts are 5 per mill, and 10 percent of sawdust is added for adjusting the water content of the materials. Setting an index of equipment parameters: setting parameters of a heating plate at 50 ℃ and a biochemical bin at 60 ℃ after materials are added into a reactor, and stirring parameters: forward rotation for 20min, reverse rotation for 20min, and stop for 20min; after 2h incubation, the running program was adjusted to a heating plate temperature of 100 ℃, a biochemical bin temperature of 105 ℃, and stirring parameters: forward rotation for 20min, reverse rotation for 20min, and stop for 20min; the aeration and air suction interval is set to be 50-55 ℃, the operation is continued for 72 hours, the sampling is continuously carried out at 16:00 a day, and the water content, pH, GI and power consumption of the kitchen waste are measured, as shown in figures 3-4.
As can be seen from FIG. 3, the initial water content of each treatment material is about 79%, and after 3 days of biological drying treatment, the water contents are 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 decrease of the VH1 (M) bacteria added was most remarkable. Of the three treatments, the composite microbial inoculum obtained in example 2 was added with the lowest power consumption. The later stage may be due to lower power consumption caused by factors such as lower water content of the material, increased water transmission efficiency, and increased capability of maintaining temperature of microbial decomposition exotherm. It can be seen that the higher the water content, the higher the energy consumption required.
As can be seen from fig. 4, the pH of the kitchen waste added with VH1 (M) rises rapidly, but the pH change of each treatment is basically 5-5.4, probably because acidic substances are decomposed by microorganisms, the pH slightly rises, the biological drying rate is too high, the water content drops too rapidly, the heap environment is not suitable for microorganism growth, and the pH does not rise to 7.0 or above. In the whole biological drying process, the germination rate is more than 80 after being treated by adding the VH1 (M) microbial inoculum, thereby meeting the requirement of decomposition.
It will be appreciated by persons skilled in the art that the foregoing description is a preferred embodiment of the invention, and is not intended to limit the invention, but rather to limit the invention to the specific embodiments described, and that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for elements thereof, for the purposes of those skilled in the art. Modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (3)
1. The thermophilic composite microbial inoculant for the aerobic fermentation of organic waste is characterized by comprising the following components:
15 to 20vol% of streptococcus thermophilus
20 to 25vol percent of bacillus subtilis
Bacillus licheniformis 20vol%
Bacillus thermodenitrificans 20vol%
20vol% of Brevibacillus bordetention;
wherein, bacillus licheniformisBacillus licheniformis) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation date of 2020, 11 months and 26 days and a preservation number of CGMCC No.21231;
brevibacillus borsteense @Brevibacillus borstelensis) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation date of 2020, 11 months and 26 days and a preservation number of CGMCC No.21232.
2. The thermophilic composite microbial inoculant for aerobic fermentation of organic waste according to claim 1, wherein the volume ratio of streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus thermodenitrificans and Brevibacillus borstele is 15vol%, 25vol%, 20vol% and 20vol%.
3. The thermophilic composite microbial inoculant for aerobic fermentation of organic waste according to claim 1, wherein the volume ratio of streptococcus thermophilus, bacillus subtilis, bacillus licheniformis, bacillus thermodenitrificans and Brevibacillus borstele is 20vol%, 20vol%.
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