CN114149944B - Microorganism combination capable of efficiently adsorbing malodorous gas at tail end, enhanced colonization and application - Google Patents
Microorganism combination capable of efficiently adsorbing malodorous gas at tail end, enhanced colonization and application Download PDFInfo
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- 244000005700 microbiome Species 0.000 title claims abstract description 70
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- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 33
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- 241000235645 Pichia kudriavzevii Species 0.000 description 6
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- 238000007796 conventional method Methods 0.000 description 4
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Classifications
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/02—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols by adsorption, e.g. preparative gas chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses a microorganism combination and enhanced colonization of a terminal for efficiently adsorbing malodorous gas and application thereof, and relates to the technical field of gas purification. The odor adsorption microorganisms comprise pichia pastoris, bacillus subtilis and paracoccus denitrificans, are derived from natural biomass fermentation, can ensure the biological safety of the microorganisms, respond to the concept of environmental protection and emission reduction, and have wide application range. The invention provides a method for strengthening colonization and odor adsorption by using biochar combined by microorganisms, thereby achieving the purposes of promoting microorganism reproduction and inhibiting odor generation. The functions of the microorganisms in the composite flora are complementary, and the microorganisms can be self-propagated in the adsorption device, so that the frequency of replacement of the adsorption component is reduced, and the high efficiency and economical efficiency of odor adsorption are ensured.
Description
Technical Field
The invention belongs to the technical field of gas purification, and particularly relates to a microorganism combination capable of efficiently adsorbing malodorous gas at the tail end, enhanced colonization and application.
Background
The mass production of malodorous gases seriously affects the living environment of residents and endangers the physical health of human beings. The livestock manure and kitchen waste are two types of biomass which are easy to decompose and produce odor, the daily discharge amount of the livestock manure in China reaches 700 ten thousand tons at present, the annual production total amount is about 24 hundred million tons, and the production amount of the excrement is about 16 hundred million tons and 8 hundred million tons respectively; in the urban household garbage structure of China, the kitchen garbage accounts for 30% -50%, the generation amount of the urban kitchen garbage is not less than 6000 ten thousand tons every year, and the generation amount also shows a continuously increasing trend. The main components of the livestock manure and kitchen garbage are organic matters such as protein, sugar, fat and the like, wherein the organic matters such as protein, amino acid and the like can perform decarboxylation and deamination due to the activity of microorganisms, so that a large amount of malodorous gas is generated, and the air quality and the body health of surrounding residents are seriously influenced; thus, the treatment of the odor generated by the decomposition of the biomass is not slow.
At present, foreign countries are in the leading position in the development of microbial deodorant products, but high-efficiency deodorant products with independent intellectual property rights in China are few, and the defects of poor adaptability, poor effect and the like exist in practical application, so that development work is urgently needed in the aspects of adsorption carriers, enhanced colonization, high-efficiency deodorant strain screening and the like, the targeted requirements of treatment of various pollution gas types such as industrial and agricultural production, living emission and the like are met, and the effective treatment of malodorous gas is realized.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a group of microorganism combinations with complementary functions and a preparation method of an immobilized odor adsorption microbial agent.
The technical method of the invention is as follows:
the invention provides a microorganism combination with a tail end capable of efficiently adsorbing malodorous gas, wherein the microorganism combination comprises one or more of pichia pastoris, bacillus subtilis or paracoccus denitrificans; when the three are combined, the mass ratio of the bacterial bodies of the pichia pastoris, the bacillus subtilis and the paracoccus denitrificans is (1-3) to (1-3).
The strain is derived from decomposed natural biomass, has high safety, wherein pichia pastoris is Pichia kudriavzevii NAU-OR2, is classified and named Pichia kudriavzevii, and is preserved in the microorganism strain preservation center of Guangdong province at 8/31 of 2021, and addresses: building 5, no. 59 of the university of Mitsui 100, guangzhou City, accession number GDMCC NO:61876, bacillus subtilis Bacillus subtilis NAU-OR4, classified under the name Bacillus subtilis, accession number of microorganism strain collection, guangdong province, 8/18, 2021, address: building 5, no. 59 of the university of Mitsui 100, guangzhou City, accession number GDMCC NO:61878, paracoccus denitrificans Paracoccus denitrificans NAU-OR1, classified and named Paracoccus denitrificans, deposited at the microorganism seed collection, cantonese province, 10 months 28, 2021, address: building 5, no. 59 of Guangzhou Xian Zhonglu 100 college, with the preservation number of GDMCC No. 61875.
In some embodiments, the mass ratio of the bacterial cells of the pichia pastoris, the bacillus subtilis and the paracoccus denitrificans is (1-2): 2-3): 1-3.
The invention also provides a preparation method of the odor-inhibiting microorganism combination, which comprises the steps of mixing strains with a certain proportion; the preparation method of each strain may include the following steps in a specific example, according to a conventional method in the art: respectively activating each strain stored at-80 ℃ on a flat culture medium, then picking a single colony, culturing in a liquid culture medium to a logarithmic phase, transferring into a seed culture medium, and performing gradual expansion culture; and centrifugally collecting thalli and spores. During the expansion culture, components favorable for the spore production of the thalli can be added.
The invention also provides a deodorizing microbial agent, which is prepared by uniformly mixing the microbial combination, the biochar and the colonial fluid, and incubating for 20-25 hours at 28-32 ℃.
In one embodiment, the ratio of biochar, microorganism combination and colonisation solution is 1g (2-3) g (0.5-1.5) ml; in some specific examples, the ratio of biochar, microorganism combination and colonising solution is 1g to 2g to 1ml.
In one embodiment, the mixture ratio of the colonising solution is as follows: 0.5 to 1.5g/L of monopotassium phosphate, 0.5 to 1.5g/L of disodium hydrogen phosphate, 0.05 to 0.15g/L of calcium chloride, 0.5 to 1.5g/L of ammonium chloride, 0.5 to 1.5g/L of soluble starch, 0.005 to 0.015g/L of zinc sulfate, 0.005 to 0.015g/L of copper sulfate, 0.05 to 0.07g/L of magnesium sulfate heptahydrate and pH value of 6 to 8.
The invention also provides an application of the deodorizing microorganism combination or the deodorizing microbial inoculum in treating malodorous gas produced by biomass fermentation.
The biomass of the invention can be any substance capable of generating ammonia, hydrogen sulfide, mercaptan, thioether, volatile organic compounds, comprehensive malodor and other gases, and in a specific example, the malodor-generating biomass by fermentation is livestock manure or kitchen waste. The malodorous gas can be one or more of common malodorous gas such as ammonia, hydrogen sulfide, mercaptan, thioether, volatile organic compounds or comprehensive malodorous gas.
Preferably, for odor adsorption of livestock manure with high ammonia and hydrogen sulfide yield, the mass ratio of the pichia pastoris, the bacillus subtilis and the paracoccus denitrificans is 2:2:1;
preferably, for odor adsorption of kitchen waste with low yield of ammonia and hydrogen sulfide and relatively high odor concentration, the mass ratio of the pichia pastoris, the bacillus subtilis and the paracoccus denitrificans is 3:1:1.
In some specific application examples, the malodorous gas is passed through a carrier containing a combination of microorganisms or a deodorant, and the carrier may be a liquid containing a combination of microorganisms, a tubular container containing a deodorant, or the like.
The invention has the advantages over the prior art: the functions among the strains are complementary, and the compound deodorization effect is obvious; the compound microbial inoculum is loaded on the biochar, so that the deodorizing efficiency is high, the survival time of each strain is long, and the compound microbial inoculum can be repeatedly and efficiently used for a long time.
The invention provides a high-efficiency odor adsorption microorganism combination, which is a novel biological odor adsorbent prepared by fermenting and compounding microorganism strains, wherein the effective components of the microorganism combination are pichia pastoris, bacillus subtilis and paracoccus denitrificans are derived from kitchen waste or livestock manure. The screened microorganisms come from living biomass waste, so that the biological safety of the living biomass waste can be ensured; and the odor adsorption bacteria can adsorb and convert components in odor, thereby reducing the emission of malodorous gas and well responding to the concept of environmental protection and emission reduction. The odor adsorption microorganism combination can be widely applied to places with odor such as livestock farms, garbage transfer stations, garbage disposal sites, zoos, sewage treatment plants, sewage pools (ditches), toilets, restaurants and the like. The microbial composition can be used simply and conveniently through a pipeline or liquid containing the microbial composition.
The preparation and use method of the odor adsorption microorganism combination provided by the invention not only can greatly improve the action effect, but also can furthest reduce the treatment cost, thereby being beneficial to large-area popularization and application.
In the invention, when the microorganism combination is used for odor adsorption, the process of charcoal and microorganism 'filtering' is involved, and odor related substances are converted or degraded through metabolic activity of microorganisms based on odor absorption and microorganism fixation conversion, wherein the specific process is as follows: (1) The odor molecules are fixed in the pores through adsorption of biochar; (2) The immobilized odor molecules are dissolved into the colonisation liquid and enter into cells of the microorganism combination; (3) After entering cells, the odor is decomposed and utilized by microorganisms as a nutrient in the body, so that the odor is removed. The biodegradation of malodorous substances is the speed limiting stage of the process, and the metabolites generated after the malodorous substances are digested and absorbed by the microorganisms are further used as nutrients of other microorganisms to continue the absorption and digestion, so that the malodorous substances are degraded gradually by circulation.
Drawings
FIG. 1 is a graph showing the change of odor release of livestock and poultry feces with time;
FIG. 2 is a characteristic of the odor release of kitchen waste with time;
FIG. 3 shows the adsorption removal of odor components by three strains of microorganisms;
FIG. 4 shows the effect of different proportions on odor adsorption removal generated by livestock manure fermentation;
FIG. 5 is a graph showing the effect of enhanced colonisation of odor adsorbing microorganism combination propagation;
FIG. 6 shows the effect of various combinations of biochar microorganisms on odor adsorption.
Detailed Description
The following examples are provided to illustrate the combination of microorganisms and methods of use of the invention for efficiently inhibiting the production of malodor by fermentation of biomass at a source, but are not to be construed as limiting the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The research of the invention finds that the comprehensive odor concentration of the livestock manure after fermentation can be rapidly increased in the initial stage, and the comprehensive odor concentration reaches 424.98 (dimensionless, according to per gram of dry manure, the following is the same); at 72h, peak 493.01 is reached; at 240h, the comprehensive odor concentration is reduced to 267.85, and the concentration is different from the concentration of each measuring point of 48-168 h, but the comprehensive odor concentration is maintained at a higher level, and has the characteristic of continuous release; the ammonia gas is less released in the early stage, is greatly improved in the later stage, and is continuously in the vicinity of the peak value from 96 to 168 hours; the release of hydrogen sulfide, methyl mercaptan, volatile organic compounds, etc. tends to be low in the early stage and to decrease after reaching the peak, and the persistence is not strong (fig. 1). The odor yield after the kitchen waste fermentation is about 1 order of magnitude less than that of livestock manure, wherein the comprehensive malodor is less released in the initial stage, reaches a peak value 44.37 (according to the dry kitchen waste per gram, the same applies hereinafter) at 120 hours, then falls to 4.40, and the kitchen waste is lower in the early stage of odor production, and falls after reaching the maximum peak value, so that the persistence is not strong; the ammonia gas is released higher in the early stage, and is slightly lifted in the later stage; the release of methyl mercaptan, volatile organic compounds and the like has the tendency of lower earlier stage and decline after reaching a peak value, and the persistence is not strong; the amount of hydrogen sulfide released was very small (fig. 2).
Unless otherwise indicated, the media used in the examples below were as follows:
the liquid culture medium or seed culture medium of pichia pastoris comprises the following components: 10g/L of yeast extract, 20g/L of bactopeptone, 20g/L of agar, 200g/L of potato and 20g/L of glucose;
the liquid culture medium or seed culture medium of the bacillus subtilis comprises the following components: 10g/L tryptone, 5g/L yeast extract, 5g/L sodium chloride and 1N sodium hydroxide 1mL/L;
the liquid culture medium or seed culture medium of the paracoccus denitrificans comprises the following components: 5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 0.7g/L of monopotassium phosphate and 0.5g/L of calcium chloride.
EXAMPLE 1 isolation, identification and preservation of three strains
The pichia pastoris, the bacillus subtilis and the paracoccus denitrificans are all separated from the livestock manure of primary fermentation, 10.0g (wet weight) of the livestock manure is added into 20mL of water, and the mixture is stirred uniformly; after a little precipitation, 100 mu L of supernatant is respectively coated on YPD, LB and paracoccus denitrificans culture medium plates, colonies are selected for purification culture after growing out, functional strains are subjected to function verification and are subjected to sample feeding, sequencing and identification, wherein the ITS sequence of Pichia pastoris (Pichia kudriavzevii) NAU-OR2 is shown as SEQ ID NO.1, the strains are determined to be Pichia pastoris (Pichia kudriavzevii) after comparison, the classification is Pichia kudriavzevii, and the strains are deposited in the microorganism strain collection center of Guangdong province on 8 months 31 of 2021, and the number is: GDMCC NO. 61876. Bacillus subtilis is Bacillus subtilis NAU-OR4, 16sRNA is shown in SEQ ID NO.2, and is classified and named Bacillus subtilis, and is deposited in the microorganism strain collection of Guangdong province at 8.18 of 2021, address: building 5, no. 59 of Guangzhou Xian Zhonglu 100, china, with the preservation number of GDMCC NO. 61878. Paracoccus denitrificans is Paracoccus denitrificans NAU-OR1, 16sRNA is shown in SEQ ID NO.3, classified and named Paracoccus denitrificans, and deposited at the microorganism strain collection of Guangdong province at 10/28 of 2021, address: building 5, no. 59 of Guangzhou Xian Zhonglu 100 college, with the preservation number of GDMCC No. 61875.
EXAMPLE 2 preparation of odor adsorbing microorganism combinations
The preparation method of each strain comprises the following steps: activating the strain stored at-80 ℃ on a solid culture medium, picking a single colony, culturing in a liquid culture medium to a logarithmic phase, transferring into a seed culture medium, and performing expansion culture step by step; centrifugally collecting thalli and spores; adding components beneficial to spore production of the thalli during the expansion culture.
According to the method, after strains of pichia pastoris, paracoccus denitrificans and bacillus subtilis are respectively activated on a solid culture medium, single bacterial colonies are selected and inoculated into 2mL of corresponding liquid culture medium, after the culture is carried out to a logarithmic phase, the strains are transferred into a seed culture medium (the ratio is 1:1000), and the strains are gradually expanded to be cultured; and centrifugally collecting thalli and spores. The activated solid culture medium of the pichia pastoris comprises the following components: 10g/L of yeast extract, 20g/L of bactopeptone, 20g/L of agar, 200g/L of potato, 20g/L of glucose and 18g/L of agar; the activated solid medium of the paracoccus denitrificans comprises the following components: 5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 0.7g/L of monopotassium phosphate, 0.5g/L of calcium chloride and 18g/L of agar; the activated solid culture medium of the bacillus subtilis comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride, 1mL/L of 1N sodium hydroxide and 15g/L of agar; when the microbial inoculum is produced in large tanks, the carbon source and the nitrogen source are required to be adjusted into starch, soybean powder, amylase and the like (can be adjusted according to the conventional method in the field), and an antifoaming agent is added; to promote sporulation of the cells, an amount of molasses is added (the amount added may be adjusted according to a conventional method in the art). The culture temperature of the strain is 30-35 ℃. And centrifugally collecting thalli and spores to obtain corresponding strains.
The bacterial strains are mixed according to the proportion to form the odor adsorption microorganism combination, for example, the mass ratio of the bacterial cells of pichia pastoris, bacillus subtilis and paracoccus denitrificans of the high-efficiency odor adsorption microorganism combination is (1-3) 1-3. .
In the embodiment, the combination of odor adsorption microorganisms can strengthen the colonization on the biochar, and is filled into the steel pipe, and the mixing proportion and the strengthening colonization method of the biochar are as follows: 2g of charcoal per gram of charcoal are combined with microorganisms (wet basis) and 1mL of colonisation solution are mixed well and incubated for 24 hours in a 30℃incubator.
The microbial combination can be colonized on charcoal by adding a certain colonizing liquid, wherein the colonizing liquid comprises the following components of adding 0.1g of monopotassium phosphate, 0.1g of disodium hydrogen phosphate, 0.01g of calcium chloride, 0.1g of ammonium chloride, 0.1g of soluble starch, 0.001g of zinc sulfate, 0.001g of copper sulfate and 0.006g of magnesium sulfate heptahydrate into every 100mL of water, regulating the pH to 7, heating to fully dissolve, and cooling to 30 ℃.
Example 3: odor adsorption effect of microorganism combination
1. Materials and methods
The instrumentation used is shown in table 1.
Table 1 instrument and apparatus
Chemical reagent: disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and soluble starch were purchased from national pharmaceutical chemicals, inc; potassium nitrate and ammonium sulfate are analytically pure and purchased from Shanghai Lingfeng chemical reagent Co. Pig manure was obtained from Rugao Huari modern farm and kitchen waste was obtained from Nanjing university Pukou school canteen.
2. Test method
2.1 cell collection of cells
Activating according to the method described in example 2, picking single colonies of activated pichia pastoris, paracoccus denitrificans and bacillus subtilis, inoculating to 2mL of corresponding liquid culture medium, placing in a 30 ℃ incubator, and shaking 120r/min overnight for culture; 300 mu L of the culture solution is inoculated into 300mL of liquid culture medium, 160r/min is used for culturing to logarithmic phase, the time for culturing pichia pastoris and bacillus subtilis is 6 hours, and the time for culturing paracoccus denitrificans is 24 hours. And centrifuging for 10min under 10000r/min to collect thalli.
2.2 determination of odor adsorption Effect
Fermenting livestock and poultry manure or kitchen garbage in a certain container to generate odor, and respectively collecting the odor by using a 2L gas collecting bag; uniformly mixing different gas collecting bags into a large gas collecting bag, and measuring and recording odor parameters (A); ammonia, hydrogen sulfide, methyl mercaptan, volatile and comprehensive malodorous gas production of the pre-treatment pig manure are 19.05 ppm/gram dry manure, 3.17 ppm/gram dry manure, 3.75 ppm/gram dry manure, 2.31 ppm/gram dry manure and 659.78/gram dry manure, respectively; the untreated kitchen waste has the yields of ammonia gas, hydrogen sulfide, methyl mercaptan, volatile and comprehensive malodorous gases of 3.66 ppm/g dry manure, 0.03 ppm/g dry manure, 1.98 ppm/g dry manure, 1.72 ppm/g dry manure and 97.10/g dry waste respectively. Preparing bacterial liquid from the bacterial cells collected in 2.1 and water according to a ratio of 1:100, and measuring each parameter (B) after odor passes through the bacterial liquid by taking water as a reference, so as to obtain the reduction rate and the reduction capability of each bacterial strain on odor indexes; the calculation formula of the reduction rate (Y): y= (a-B)/a×100%; the calculation formula of the curtailment capability (N): n=y Bacteria (fungus) /Y Water and its preparation method 。
2.3 experiments on the combined effects of microorganisms
Pichia pastoris, paracoccus denitrificans and bacillus subtilis are combined according to the mass (wet basis) ratio of 1:1:1, 2:1:1, 3:1:1, 2:2:1, 1:3:1, 1:3:2 and 1:3:3 of the bacteria, and are prepared into bacterial liquid according to 1:100 with water, 1L of odor (with the same concentration as before) in an air collecting bag passes through 500mL of bacterial liquid, odor indexes before and after passing are measured, and the cutting rate and the cutting capacity of each combination are calculated.
2.4 experiments on the effects of different ratios of biochar and microbial inoculant
Biochar (dry basis), microorganism (wet basis) and colonisation liquid (mL) are prepared into a combined odor adsorbent according to the method of example 2 according to the ratio of 3:1:3, 2:1:2, 1:1:1, 1:2:1 and 1:3:1, and are filled into a steel pipe with the inner diameter of 5cm, and two ends are wrapped by 3 layers of gauze; 1L of odor (the concentration is the same as the previous) in the gas collecting bag is introduced into a steel pipe filled with the adsorbent, the change of the quantity before and after the odor is introduced is recorded, and the odor reduction rate is calculated.
2.5 data processing and analysis
The resulting data were statistically analyzed using IBM SPSS Statistics, excel 2016, origin 2018 software; the difference in significance between the different treatments was examined using Duncan, α=0.05.
3. Experimental results
3.1 odor adsorption Effect of three microorganisms
The study selects 6 strains of lactobacillus, pichia pastoris (GDMCC NO: 61876), bacillus subtilis (GDMCC NO: 61878), paracoccus denitrificans (GDMCC NO: 61875), saccharomyces cerevisiae, bacillus licheniformis and the like, and takes water as a control, and the result shows that the selected pichia pastoris, paracoccus denitrificans and bacillus subtilis have higher reduction rates on ammonia, hydrogen sulfide and comprehensive malodor (figure 3), wherein the reduction rates of ammonia are 85.91%, 68.03% and 69.69%, and the reduction capacities are 2.35, 1.88 and 1.91. The selected bacillus subtilis has the strongest hydrogen sulfide removal capability, the hydrogen sulfide reduction rate reaches 90.80 percent, and the reduction capability is 1.83; the hydrogen sulfide reduction rates of pichia pastoris and paracoccus denitrificans also reach 89.8% and 81.48%, respectively, and the reduction capacities are 1.81 and 1.65. The reduction rates of the selected pichia pastoris, bacillus subtilis and paracoccus denitrificans on comprehensive malodor reach 90.01%, 72.9% and 72.2%, and the reduction capacities are 3.42, 2.77 and 2.73 respectively; the nitrifying bacteria, the saccharomycetes and the decomposing bacteria have good malodor removing effect.
3.2 influence of microorganism ratio on odor adsorption Effect
Pichia pastoris, paracoccus denitrificans and bacillus subtilis are combined according to the mass (wet basis) ratio of 1:1:1, 2:1:1, 3:1:1, 2:2:1, 1:3:1, 1:3:2 and 1:3:3 of the bacteria, and are prepared into bacterial liquid according to the ratio of 1:100 with water, and the odor in the air collecting bag is passed through the bacterial liquid. For malodorous gas generated by fermentation of livestock manure, the range of the invention has good malodorous adsorption effect, and particularly, the malodorous adsorption effect of pichia pastoris, bacillus subtilis and paracoccus denitrificans with the mass ratio of 2:2:1 is obviously better than that of other ratios (shown in figure 4, P < 0.05); and when the quality of the odor generated by the fermentation of the adsorbed kitchen waste is adjusted to be 3:1:1, the odor adsorbing agent has the best effect.
3.3 Effect of colonising solution on odor adsorbing microorganism combinations
As can be seen from FIG. 5, when odor adsorbing microorganisms were immobilized on biochar, after 1mL of the colonisation solution was added or no colonisation solution was added to each gram of biochar, the colony of microorganisms was changed by incubation at 30℃for 30 hours, and as a result, it was found that the increase in colonisation solution was 4.2-fold within 6 hours after the addition of the colonisation solution, but only 0.2-fold was obtained when no colonisation solution was added, and the increase in colony count was not significant when no colonisation solution was added during the whole observation period (FIG. 5), and it was seen that the colonisation solution according to the present invention could promote the increase in microorganisms.
3.4 influence of the ratio of the biochar microbial inoculant on the odor adsorption effect
The biochar (dry basis mass), the microorganism (wet basis mass) and the colonisation liquid (mL) are prepared into a combined odor adsorbent according to the ratio of 3:1:3, 2:1:2, 1:1:1, 1:2:1 and 1:3:1, and are filled into a steel pipe with the inner diameter of 5cm, and the result is shown in figure 6, when the ratio of the three is found to be 1:2:1, the removal rate of ammonia, hydrogen sulfide and comprehensive malodor is obviously higher than that of 3:1:3, 2:1:2 and 1:1:1; there is no significant difference between 1:2:1 and 1:3:1, and from the economical point of view, 1:2:1 is the optimal ratio.
Example 4: adsorption of pig manure odor
Pichia pastoris, bacillus subtilis and paracoccus denitrificans are proportioned according to a mass ratio of 2:2:1 of bacteria, 2g (wet basis) of a combination of each gram of biochar and microorganisms and 1mL of a colonisation liquid are uniformly mixed, and after incubation for 24 hours in a 30 ℃ incubator, the mixture is used as a filler to be filled into a steel pipe, and is connected into a Huari modern farm odor pumping and exhausting system for absorbing the odor of a live pig farm, and as a result, the pig manure ammonia gas, hydrogen sulfide and comprehensive malodorous gas yield are respectively 19.05 ppm/gram of dry manure, 3.17 ppm/gram of dry manure and 659.78/gram of dry manure before the odor of the manure is untreated. After suction and exhaust adsorption, ammonia, hydrogen sulfide and comprehensive malodor are reduced by more than 90%, the culture environment is greatly purified, and adverse effects on surrounding residents are reduced.
Example 5: adsorption of odor of leftovers of canteen
In this test, three plastic bottles (numbered #1, #2 and # 3) of the same volume were filled with 2kg of the remaining meal in the mixing canteen, and the mouth was closed to allow the bottles to ferment at 35℃and accumulate odor.
Two bottles #1 and #2 are opened after half a month, and malodor smell exists in the two bottles. Connecting the #1 bottle with a steel pipe filled with the combined odor adsorbent; the filled adsorbent consists of biochar (dry basis mass), microorganisms (wet basis mass) and colonisation liquid (mL) according to the proportion of 1:2:1, and the mass ratio of the pichia pastoris, bacillus subtilis and paracoccus denitrificans in the microorganism combination is 3:1:1. At the subsequent 24 hours, the odor in the #1 bottle was significantly lighter and the outer vent was also free of significant odor; no significant change in malodor was seen in bottle No. 2. Continuously connecting the steel pipe filled with the odor adsorbent with the #1, and regularly pumping and exhausting for 10 minutes every day; the #2 bottle was capped and placed on hold.
After one month, three bottles were opened and the odor of #1, #2, #3 was compared. The odor indexes of ammonia gas, hydrogen sulfide, comprehensive malodor and the like in the bottle #1 are lower than those of the two bottles #2 and #3 by more than 95%, and no obvious difference exists between the odor indexes of the two bottles #2 and # 3. Through the experiment, the microorganism combination and biochar combined adsorbent has obvious adsorption effect on odor and has lasting effect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Nanjing agricultural university
<120> microorganism combination for efficiently adsorbing malodorous gas at terminal, enhanced colonisation and application
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ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 60
cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtctga 120
accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 180
cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 240
ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 300
gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 360
cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 420
gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 480
gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 540
gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 600
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 660
agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 720
aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 780
tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 840
aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 960
gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1080
tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1140
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1200
aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1260
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cgcgtgagtg atgaaggccc tagggttgta aagctctttc agctgggaag ataatgacgg 360
taccagcaaa agaagccccg gctaactccg tgccagcagc cgcggtaata cggagggggc 420
tagcgttgtt cggaattact gggcgtaaag cgcacgtagg cggaccggaa agttgggggt 480
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cgaaggcggc tcactggctc gatactgacg ctgaggtgcg aaagcgtggg gagcaaacag 660
gattagatac cctggtagtc cacgccgtaa acgatgaatg ccagtcgtcg ggcagcatgc 720
cgttcggtga cacacctaac ggattaagca ttccgcctgg ggagtacggt cgcaagatta 780
aaactcaaag gaattggcgg ggggccgcac aagcggtgga gcatgtggtt taattcgaag 840
ctacgcgcag aaccttacca tcccttgaca tcgcaggccc gctccagaga tggagttttg 900
ttgtaagagg cctgtggtca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt 960
cggttaagtc ctgcaacgag cgcacccccc actgttagtt gccagcattt ggttgggcac 1020
tataagagaa ctgccgatga taagtcggag gaaggtgtgg atgacgtcaa gtcctcatgg 1080
cccttacggg ttgggctaca cacgtgctac aatggtggtg acagtgggtt aatccccaaa 1140
agccatctca gttcggattg gggtctgcaa ctcgacccca tgaagttgga atcgctagta 1200
atcgcggaac agcatgccgc ggtgaatacg ttcccgggcc ctgtacacac cccccgtcac 1260
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Claims (11)
1. The microbial combination is characterized by comprising pichia pastoris, bacillus subtilis and paracoccus denitrificans; the mass ratio of the bacterial bodies of the pichia pastoris, the bacillus subtilis and the paracoccus denitrificans is (1-3) to (1-3); the Pichia pastoris isPichia kudriavzevii NAU-OR2, accession number GDMCC NO 61876, bacillus subtilisBacillus subtilisNAU-OR4, accession number GDMCC NO 61878, paracoccus denitrificansParacoccus denitrificansNAU-OR1, accession number GDMCC No. 61875.
2. The microbial combination of claim 1, wherein the mass ratio of the bacterial cells of pichia pastoris, bacillus subtilis and paracoccus denitrificans is (1-2): 2-3): 1-3.
3. The deodorant bacterial agent is characterized in that the deodorant bacterial agent is prepared by uniformly mixing the microorganism combination, the biochar and the colonial fluid according to claim 1, and incubating the mixture at 28-32 ℃ for 20-25 hours, wherein the colonial fluid comprises the following components: potassium dihydrogen phosphate 0.5-1.5 g/L, disodium hydrogen phosphate 0.5-1.5 g/L, calcium chloride 0.05-0.15 g/L, ammonium chloride 0.5-1.5 g/L, soluble starch 0.5-1.5 g/L, zinc sulfate 0.005-0.015 g/L, copper sulfate 0.005-0.015 g/L, magnesium sulfate heptahydrate 0.05-0.07 g/L, and pH 6-8.
4. The deodorant bacterial agent according to claim 3, wherein the ratio of the amount of the biochar, the microorganism combination and the colonising liquid is 1g (2-3 g) (0.5-1.5 ml).
5. The deodorant bacterial agent according to claim 4, wherein the ratio of the amount of the charcoal, the microorganism combination and the colonisation liquid is 1 g/2 g/1 ml.
6. Use of the microorganism combination of claim 1 or2 or the deodorant bacterial agent of any one of claims 3 to 5 for the treatment of malodorous gases produced by biomass fermentation.
7. The use according to claim 6, wherein the biomass is livestock manure or kitchen waste; the malodorous gas is one or more of ammonia gas, hydrogen sulfide, mercaptan, thioether, volatile organic compounds or comprehensive malodorous gas.
8. The use according to claim 7, wherein when the biomass is livestock manure, the mass ratio of the cells of pichia pastoris, bacillus subtilis and paracoccus denitrificans is 2:2:1.
9. The use according to claim 7, wherein when the biomass is kitchen waste, the mass ratio of the cells of pichia pastoris, bacillus subtilis and paracoccus denitrificans is 3:1:1.
10. The use according to any one of claims 6 to 9, wherein the use is by passing malodorous gases through a carrier containing a combination of microorganisms or a deodorizing agent.
11. Use according to claim 10, wherein the carrier is a liquid containing a combination of microorganisms or a tubular container containing a deodorant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202111447845.1A CN114149944B (en) | 2021-11-30 | 2021-11-30 | Microorganism combination capable of efficiently adsorbing malodorous gas at tail end, enhanced colonization and application |
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| CN104611258A (en) * | 2014-12-30 | 2015-05-13 | 青岛蔚蓝天成生物科技有限公司 | Bacillus subtilis and biological deodorant containing bacterial strain of bacillus subtilis |
| CN110117567A (en) * | 2019-06-04 | 2019-08-13 | 西安工程大学 | A kind of Paracoccus denitrificans bacterial strain screening and its application in deodorization |
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| CN104611258A (en) * | 2014-12-30 | 2015-05-13 | 青岛蔚蓝天成生物科技有限公司 | Bacillus subtilis and biological deodorant containing bacterial strain of bacillus subtilis |
| CN110358699A (en) * | 2019-05-10 | 2019-10-22 | 基因赛奥(大连)生物科技发展有限公司 | A kind of complex microorganism deodorizing microorganism and the preparation method and application thereof |
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