CN111117905B

CN111117905B - Pichia mansoni

Info

Publication number: CN111117905B
Application number: CN202010093473.6A
Authority: CN
Inventors: 陈贯虹; 黄玉杰; 张强; 王加宁; 高永超; 张闻; 王磊磊; 赵庆庆; 郑立稳
Original assignee: Ecology Institute Shandong Academy Of Sciences
Current assignee: Ecology Institute Shandong Academy Of Sciences
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2022-03-22
Anticipated expiration: 2038-12-18
Also published as: CN109609401A; CN111172078A; CN111172078B; CN109609401B; CN111117905A

Abstract

The invention relates to a composite microbial deodorant bacterial agent and a preparation method and application thereof, wherein the bacterial agent comprises 2-2 of Bacillus cereus, 2-6 of Pichia manshurica and 2-20 of Lactobacillus zeae, and the total bacterial number of the composite microbial deodorant bacterial agent is more than or equal to 2 multiplied by 10⁹one/mL. According to the invention, through mixing three strains of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20, antagonistic reaction among the three strains is avoided, and NH removal can be obviously promoted₃And H₂According to the effect of S, actual detection shows that the ammonia gas removal rate of the microbial deodorant agent compounded by the three strains reaches 73.04%, and the hydrogen sulfide removal rate reaches 71.19%.

Description

Pichia mansoni

The present application is a divisional application of the following applications: application date: 12/18/2018, application No.: 2018115479500, title of invention: a composite microbial deodorant, and its preparation method and application are provided.

Technical Field

The invention relates to a composite microbial deodorant bacterial agent, a preparation method and application thereof, and belongs to the technical field of harmless treatment or biological deodorization of livestock and poultry manure.

Background

With the continuous development of livestock and poultry industry in China, the intensification degree is higher and higher, the livestock and poultry breeding quantity is increased dramatically, the discharge amount of excrement is increased, and certain pollution is caused to air, water and soil. Among them, the malodorous pollution is considered to be one of six public hazards next to the noise pollution, which not only seriously pollutes the environment, but also even harms the health of people, and has the dual properties of air pollution and harmful gas pollution. A large amount of livestock and poultry manure is fermented in the stacking process to generate various malodorous substances, including NH₃、H₂S, indole, methyl mercaptan and the like can cause harm to the health of people and livestock after long-term contact, and have potential possibility of chronic poisoning. It is reported that a 3-ten thousand-size egg-laying farm can discharge more than 1.8kg of NH per day₃In the air, due to the long-term contact with NH₃The eye conjunctivitis of the poultry can be caused, and the normal intake and drinking of the poultry are influenced; secondly, these malodors can also be a source of mosquito and fly growth, spread of disease, and contribute to greenhouse effect and acid rain. Thus, the ammonia gas is reducedAnd the emission of malodorous gases such as hydrogen sulfide and the like are one of the research hotspots for solving the key problems of the existing compost deodorization.

At present, the livestock and poultry manure deodorization treatment at home and abroad mainly adopts physical, chemical and biological deodorization technologies to change the object image and structure of malodorous gas so as to achieve the deodorization effect. The chemical method mainly utilizes strong oxidant potassium permanganate, hydrogen peroxide, etc. to oxidize partial odor components into less-odor and odorless substances. Physical methods include mechanical ventilation to reduce odor concentration, or absorption of odors by absorbents. The two technologies are easy to increase the cost and cannot be repeatedly used while quickly deodorizing, so that secondary pollution to the environment is caused. The biological deodorization method is a method for treating odor pollution developed in the last 50 th century, and the method utilizes physiological metabolic activity of microorganisms to degrade odor substances to convert the odor substances into odorless substances, has the characteristics of high treatment efficiency, no secondary pollution, convenience in operation, low cost and the like, and has become a main method in odor control research and application at home and abroad.

Chinese patent document CN102851210A (application No. 201210190607.1) discloses a microbial deodorant microbial inoculum of livestock and poultry manure, a preparation method and an application thereof, wherein the microbial inoculum contains a collection number of CGMCC NO: 5982 and the preservation number is CGMCC NO: 5984 Saccharomyces cerevisiae, the microbial inoculum is added into pig manure, the reduction rate of ammonia gas is 44-51%, and the reduction rate of hydrogen sulfide is 33-38%. The microbial deodorization process mainly plays roles in functions of oxidizing sulfides, heterotrophic nitrification, aerobic denitrification, reduction and the like of microorganisms, so that the key point of deodorization is screening of efficient microorganisms, and the biological characteristics and deodorization effect of the microorganisms are researched, thereby laying a foundation for preparing novel microbial deodorization microbial inoculum.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a composite microbial deodorant bacterial agent and a preparation method and application thereof.

The technical scheme of the invention is as follows:

a composite microbial deodorant bacterial agent comprises Bacillus cereus (B)2-2 parts of bacillus cereus), 2-6 parts of Pichia manshurica and 2-20 parts of Lactobacillus zeae, wherein the total bacterial number of the composite microbial deodorant bacterial agent is more than or equal to 1 multiplied by 10⁹Per mL;

the Bacillus cereus (Bacillus cereus)2-2 is preserved in the China general microbiological culture Collection center in 26 th 09.2018, and the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16527;

2-6 of the Pichia mansoni (Pichia manshurica) is preserved in the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms in 2018, 09 and 26 months, and the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16529;

the Lactobacillus zeae (Lactobacillus zeae)2-20 is preserved in the China general microbiological culture Collection center in 26.09.2018, with the preservation address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16528.

According to the invention, preferably, the bacterial number ratio of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 in the composite microbial deodorant bacterial agent is 1: (0.5-2): (0.5-2).

Further preferably, the ratio of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 in the composite microbial deodorant is 1: 1.

The preparation method of the composite microbial deodorant bacterial agent comprises the following steps:

(1) strain activation

Respectively inoculating 2-2 parts of bacillus cereus, 2-6 parts of pichia mansoni and 2-20 parts of lactobacillus zeae on corresponding liquid culture media, and culturing until the logarithmic phase of growth to obtain 2-2 seed solutions of bacillus cereus, 2-6 seed solutions of pichia mansoni and 2-20 seed solutions of lactobacillus zeae;

(2) culture of bacterial strain

Respectively inoculating the bacillus cereus 2-2 seed liquid, the pichia mansoni 2-6 seed liquid and the lactobacillus zeae 2-20 seed liquid obtained in the step (1) into corresponding amplification culture media according to the inoculation amount of 10% of the volume ratio, and performing amplification culture to obtain bacillus cereus 2-2 bacterial liquid, pichia mansoni 2-6 bacterial liquid and lactobacillus zeae 2-20 bacterial liquid;

(3) strain compounding

Detecting the bacterial counts of the bacillus cereus 2-2 bacterial liquid, the pichia mansoni 2-6 bacterial liquid and the lactobacillus zeae 2-20 bacterial liquid obtained in the step (2), and according to the ratio 1 of the bacterial counts of the bacillus cereus 2-2, the pichia mansoni 2-6 and the lactobacillus zeae 2-20: (0.5-2): (0.5-2), mixing, and adjusting the pH value to 5.0-6.5 to obtain the composite microbial deodorant bacterial agent; wherein the total bacteria number of the composite microbial deodorant bacterial agent is 1-4 multiplied by 10⁹one/mL.

Preferably, the composition of the liquid medium in step (1) is as follows:

the bacillus cereus 2-2 liquid culture medium is a nutrient broth culture medium: 10g of peptone, 3g of beef powder, 5g of sodium chloride and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2, and the components are sterilized at 121 ℃ for 20min for later use;

the liquid culture medium of Pichia mansoni 2-6 and Lactobacillus zeae 2-20 is YPD culture medium: 10g of yeast extract, 20g of glucose, 20g of peptone and 1000mL of distilled water, and sterilizing at 115 ℃ for 30min for later use.

Preferably, according to the present invention, the conditions of the culturing in step (1) are:

culturing Bacillus cereus 2-2 at 30-37 deg.C and 160rpm by shaking table until logarithmic phase of growth;

culturing Pichia mansoni 2-6 at 28-35 deg.C and 160rpm by shaking culture until logarithmic phase of growth;

the lactobacillus zeae 2-20 is cultured by shaking at 30-37 deg.C and 160rpm until the logarithmic phase of growth.

Preferably, the components of the expanding medium in the step (2) are as follows, and all the components are in percentage by mass:

bacillus cereus 2-2 expanding culture medium: 1% of glucose, 1% of corn starch, 0.2% of yeast extract and 1.5% of soybean meal;

pichia mansoniYeast 2-6 expanded Medium: 2% of glucose, 1% of yeast extract, 1% of peptone and KH₂PO₄ 0.15％，MgSO₄ 0.02％；

Lactobacillus zeae 2-20 expansion medium: 5% of brown sugar, 1% of corn starch, 1% of peptone and KH₂PO₄ 0.15％，MgSO₄ 0.02％。

Preferably, the conditions for the expanded culture in step (2) are:

culturing Bacillus cereus 2-2 at 30-37 deg.C under 160rpm for 16-24 hr;

culturing Pichia mansoni 2-6 at 28-35 deg.C under 160rpm for 24-36 hr;

culturing Lactobacillus zeae 2-20 at 30-37 deg.C and 160rpm for 20-36 hr.

According to the invention, preferably, the ratio of the number of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 in the composite microbial deodorant in step (3) is 1:1: 1.

The composite microbial deodorant bacterial agent is applied to harmless biological treatment of livestock and poultry manure.

A strain of Bacillus cereus (Bacillus cereus)2-2 is preserved in China general microbiological culture Collection center in 26 th 09.2018, with the preservation addresses as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16527.

The Bacillus cereus (Bacillus cereus)2-2 is obtained by screening from a pig farm manure pile, and bacterial colonies growing on a nutrient broth solid culture medium are milky, opaque, round or approximately round, convex, single in thallus, rod-shaped and spore-containing.

2-6 of a strain of Pichia manshurica, which is preserved in the common microorganism center of the China general microbiological culture Collection center in 2018, 09 and 26 months, wherein the preservation addresses are as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16529.

The Pichia mansoni (Pichia manshurica)2-6 is obtained by screening from pig farm manure, and the colony growing on PDA solid culture medium is white, non-transparent, round, convex, smooth in surface, oval in cell shape, suitable for growth temperature of 25-35 deg.C, and facultative anaerobic.

2-20 of a strain of Lactobacillus zeae, which is deposited in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms in 26 th 09.2018, with the deposition address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16528.

The Lactobacillus zeae (Lactobacillus zeae)2-20 is obtained by screening from a pig farm manure pile, and the bacterial colony grown in the MRS solid culture medium is milky white, round, medium in size, slightly convex, moist, neat in edge, rod-shaped in cell shape, suitable for growth at a temperature of 25-35 ℃, and facultative anaerobic.

Has the advantages that:

1. the invention obtains a strain of Bacillus cereus (Bacillus cereus)2-2 by screening, and the strain removes NH compared with the prior known Bacillus cereus₃And H₂The effect of S is obviously improved;

2. the invention obtains a strain of Pichia manshurica (Pichia manshurica)2-6 through screening, and the strain has excellent NH removal effect₃And H₂The effect of S, which is found in Pichia mansoni for the first time;

3. the Lactobacillus zeae strain 2-20 is obtained by screening, and has excellent NH removal effect₃And H₂The effect of S, which is first found in Lactobacillus zeae;

4. according to the invention, through mixing three strains of Bacillus cereus (Bacillus cereus)2-2, Pichia manshurica (Pichia manshurica)2-6 and Lactobacillus zeae (Lactobacillus zeae)2-20, the three strains can not generate antagonistic reaction, and can obviously promote and remove NH₃And H₂According to the effect of S, actual detection shows that the ammonia gas removal rate of the microbial deodorant agent compounded by the three strains reaches 73.04%, and the hydrogen sulfide removal rate reaches 71.19%.

Drawings

FIG. 1 is a photograph showing the result of PCR amplification electrophoresis of 16S rDNA strain;

in the figure: lane 1, Bacillus cereus (Bacillus cereus) 2-2; lane 2, Pichia manshurica (Pichia manshurica) 2-6; lane M, marker.

FIG. 2 is a photograph showing the result of PCR amplification electrophoresis of 26S rDNA strain;

lane 3, 26S rDNA PCR amplified fragment of Lactobacillus zeae (Lactobacillus zeae); m: marker.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.

The source of the biological material is as follows:

bacillus cereus (Bacillus cereus)2-2, which is preserved in China general microbiological culture Collection center in 26 th 09.2018, with the preservation address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16527;

pichia mansoni (Pichia manshurica)2-6, deposited in the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 26.09.2018, with the deposition address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16529;

2-20 of Lactobacillus zeae, which is preserved in the general microbiological center of the China Committee for culture Collection of microorganisms in 26 th 09.2018 with the preservation address: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16528.

Example 1: isolation of deodorizing strains

Taking 10g of manure pile outside a certain pig farm, putting into a triangular flask containing sterilized glass beads and 50ml of normal saline, shaking at 30 ℃ and 160r/min for 2h, standing for 20min, taking 1ml of supernatant, and diluting with sterilized normal water to obtain a solution with a concentration of 10^-1Continuously diluting the suspension by 10-fold gradient, sucking 200 μ l of suspension with different dilutions, spreading on MRS agar medium, nutrient broth agar medium, and YPD agar medium, and culturing at 28 deg.C for 28-And (5) observing the growth condition of the strain for 72h, and picking out a single colony for culturing. According to the strain morphology and growth condition, selecting strains with different morphologies, carrying out streak culture, and storing at low temperature. The experiment co-isolates 40 strains of bacteria, wherein 26 strains of bacteria, 8 strains of yeast and 6 strains of lactobacillus.

The culture medium and its formulation in the above examples are as follows:

MRS culture medium: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0 mL of tween, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000mL of distilled water, 6.2-6.6 of pH, and 30min of sterilization at 115 ℃ for later use. The culture medium is added with 18.0g of agar, and the other culture media are unchanged, namely the MRS solid culture medium.

Nutrient broth culture medium: 10g of peptone, 3g of beef powder, 5g of sodium chloride and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2, and the peptone is sterilized for 20min at 121 ℃ for later use. The nutrient agar culture medium is obtained by adding 18.0g of agar into the culture medium and keeping the balance of agar.

YPD medium: 10g of yeast extract, 20g of glucose, 20g of peptone and 1000mL of distilled water, and sterilizing at 115 ℃ for 30min for later use. 18.0g of agar was added to the medium, and the balance was unchanged, thus obtaining a YPD solid medium.

Example 2: screening and identification of strains

1 Primary screening of the Strain

150g of fresh pig manure was added to a 500ml Erlenmeyer flask with a stopper, the isolated strain was inoculated in an inoculum size of 5% by weight/volume, cultured in a 30 ℃ incubator for 10 days, and odor grade was evaluated by an organoleptic method, the results of which are shown in Table 1. As can be seen from Table 1, the deodorizing abilities of the isolated microorganisms are different, and comparison shows that three strains of bacteria, one strain of lactic acid bacteria and two strains of yeast have better deodorizing abilities.

Measuring the odor grade by an organoleptic method, dividing the gas odor degree by a 6-grade method:

level 0: no odor, indicated as "-";

level 1: the odor is barely felt and is indicated by "+";

and 2, stage: faint odor, indicated by "+";

and 3, level: the odor is obvious and is indicated by "+ + +";

4, level: strong odor, indicated by "+ + + +";

and 5, stage: the odor was intolerable, indicated by "+ + + +".

Table 1: preliminary screening of deodorizing strains by sensory methods

Strain numbering

Grade of odor

Strain numbering

Grade of odor

Strain numbering

Grade of odor

Strain numbering

Grade of odor

2-1

++

2-12

++

C2-1

++

C2-14

++

2-2

++

2-15

++++

C2-3

++++

C2-15

+++

2-4

+++

2-17

++++

C2-7

++++

C2-16

++++

2-6

++

2-19

+++

C2-9

+++

C2-17

++++

2-10

+++

2-20

++

C2-11

++++

C2-19

++

2 rescreening of the Strain

And (4) re-screening the strains by respectively measuring the release amount of ammonia gas and hydrogen sulfide gas.

2.1 screening of Ammonia-removing bacteria

The prepared bacterial liquid was added to a 1000ml plastic beaker with a lid containing 200g of fresh feces in a proportion of 10% and mixed well. Each large beaker was equipped with 150 mL small beaker containing 30mL boric acid absorbent for absorbing ammonia gas. Sealing, standing at 28 deg.C for 3 days, detecting ammonia release amount in fermented feces, adding sterile water with the same volume as negative control, and repeating each treatment for 3 times. And (3) measuring the ammonia release amount by taking methyl red-methylene blue as an indicator and adopting a boric acid absorption Kjeldahl method. And (4) carrying out difference significance analysis on the strain and a blank control group strain, calculating the removal rate of ammonia gas, and determining the deodorizing strain.

2.2 screening of Hydrogen sulfide-removing bacteria

The prepared bacterial liquid was added to a 1000ml plastic beaker with a lid containing 200g of fresh feces in a proportion of 10% and mixed well. Each large beaker was charged with a 50ml small beaker containing 30ml of an alkaline zinc ammine salt solution for absorption of hydrogen sulfide. Sealing, standing at 28 deg.C for 3 days, detecting the release amount of hydrogen sulfide in fermented feces, adding sterile water with the same volume as negative control, and repeating each treatment for 3 times. And (3) measuring by adopting a zinc-ammonium complex salt colorimetric method. And (4) carrying out difference significance analysis on the strain and a blank control group strain, calculating the removal rate of hydrogen sulfide, and determining the deodorizing strain.

TABLE 2 deodorizing Effect of the rescreened strains

Treatment of	2-2	2-6	2-20	C2-1	C2-13	C2-19
							Ammonia gas removal rate/%)	62.79	55.22	42.86	52.04	36.26	25.64
Hydrogen sulfide removal rate/%)	49.22	52.49	46.39	35.68	37.04	22.01

As can be seen from Table 2, strain 2-2 is on NH₃Has the best deodorization effect which reaches more than 60 percent, and the strain 2-6 is relative to NH₃And H₂The removal rate of S reaches more than 50 percent, and the best deodorization performance is shown. In addition, the strain 2-20 also shows good deodorization effect on NH₃And H₂The removal rates of S are 42.86% and 46.39%, respectively, and strain C2-1 is responsible for NH₃The removal rate is preferably 52.04%, and other strains include C2-13 and C2-19 to NH₃And H₂The removal rate of S is generally shown, so strains 2-2, 2-6 and 2-20 are selected as functional strains of the composite biological deodorant bacteria.

2.3 identification of the species of the deodorizing microorganism

2.3.1 physiological and Biochemical assays

Inoculating each obtained strain on a culture medium, culturing at 28 ℃ for 48h, observing and recording the growth condition and the morphology of colonies, observing and recording the morphology of thalli under a microscope, and carrying out physiological and biochemical tests on each strain. Comprises gram staining, catalase test, gelatin liquefaction test, indole test, VP test, starch hydrolysis test and nitrate and glucose fermentation test. The results are shown in Table 3

The bacterial colony of the strain 2-2 growing on the nutrient solid culture medium is milky white, non-transparent, round or approximately round, convex, single in thallus, rod-shaped and spore-containing.

The strain 2-6 is yeast, and the colony grown on the PDA agar plate is white, non-transparent, round, convex, smooth in surface, oval in cell shape, suitable for growth at 25-35 deg.C, and facultative anaerobic.

The bacterial strains 2 to 20 are cultured on an MRS culture medium, and found that bacterial colonies are milky white, round, medium in size, slightly convex, wet, regular in edge, rod-shaped in cell shape, suitable for growth at a temperature of 25 to 35 ℃, acidic in smell and facultative in anaerobic property.

The results of physiological and biochemical experiments with the 3 deodorizing strains obtained are shown in Table 3.

TABLE 3 physiological and biochemical characteristics of deodorizing strains

Note: "+" is positive and "-" is negative.

2.3.2 molecular validation

Respectively inoculating 2-2, 2-6 and 2-20 in nutrient broth, YPD and MRS liquid culture medium, shaking-culturing at 28 deg.C for 36h, centrifuging at 12000rpm to collect thallus, and respectively extracting 2-2,2-20 and 2-6 with Ezup column type bacterial genome DNA extraction kit and Ezup column type yeast genome DNA extraction kit. The results of PCR amplification of strains 2-2 and 2-20 using primers 7F and 1540R are shown in FIG. 1; PCR amplification of strains 2-6 with primers NL1 and NL4, results are shown in FIG. 2; the nucleotide sequence of the primer is as follows:

7F：5’-CAGAGTTTGATCCTGGCT-3’

1540R：5’-AGGAGGTGATCCAGCCGCA-3’

NL1：5’-GCATATCAATAAGCGGAGGAAAAG-3’

NL4：5’-GGTCCGTGTTTCAAGACGG-3’

and (3) purifying the PCR product, then sending the purified PCR product to Shanghai bioengineering company Limited for sequence determination, and performing homology comparison analysis on a sequencing result and a sequence in an NCBI website database. Finally, the physiological and biochemical characteristics of the three deodorization strains are combined with molecular verification, and the three strains 2-2, 2-6 and 2-20 related in the patent are respectively considered to be Bacillus cereus, Pichia manshurica and Lactobacillus zeae.

The three strains are respectively preserved, and the preservation information is as follows:

The screened strains in the patent of the invention have the effect of comparing with other deodorizing strains

The invention relates to a patent medium sieveThe selected strain Bacillus cereus (CGMCC No. 16527) is subjected to deodorization comparison experiment with the published invention patent (publication No. CN105617430A), and the microbial inoculum of the Bacillus cereus (CGMCC No. 16527) is prepared according to the preparation method of the deodorant of the group 1 embodiment in the patent CN105617430A, wherein the Bacillus cereus is not less than 3.0 multiplied by 10⁷CFU/mL, which is consistent with the cell concentration of Bacillus cereus in group 1 example of patent CN 105617430A; the microbial inoculum of the bacillus cereus CGMCC No.16527 is directly sprayed in excrement according to the method of an application experimental example in the patent CN105617430A, the experiment is repeated for three times, and the detection is carried out for 24 hours; the result shows that the deodorizing agent prepared by taking the bacillus cereus CGMCC No.16527 as the main component has the odor concentration of 55-60 dimensionless and the deodorizing effect is obviously better than that of the composite agent of the bacillus pumilus and the bacillus cereus in the 1 st group of embodiments in the patent CN 105617430A.

The strain Pichia mansonia CGMCC No.16529 screened in the patent of the invention and the published patent of the invention (publication number CN105112311A) are subjected to deodorization comparison experiment, and the fermentation of the Pichia mansonia CGMCC No.16529 is carried out according to the fermentation method of the Pichia pastoris in the specific embodiment in the patent CN 105112311A; the degradation effect of ammonia water and hydrogen sulfide of Pichia mansonia (Pichia manshurica) CGMCC No.16529 fermentation liquor is detected according to an experiment 1 method in a patent CN105112311A, and the result shows that the removal rate of the bacteria on ammonia gas is 93.5 percent, the removal rate of hydrogen sulfide is 74.2 percent, and the effect is not obviously different from the result of the experiment 1 in the patent, but no relevant report about deodorization experiments by using Pichia mansonia (Pichia manshurica) as a main component is found at present.

The patent of the aspect of deodorization by using the lactobacillus zeae is less, and the lactobacillus zeae L73 disclosed by the invention patent (publication number CN106434405A) which is disclosed at present is mainly used as an antagonistic bacterium of aeromonas hydrophila to be prepared into a bacteriostatic agent to be applied to aquaculture. In addition, in the disclosed invention patent (publication No. CN108102990A), the lactobacillus zeae is mainly used as one of the components in the microecological preparation, and the main function is to utilize the lactobacillus zeae to generate lactic acid substances and reduce the pH value of compost materials. The Lactobacillus zeae (Lactobacillus zeae) CGMCC No.16528 screened in the patent of the invention and the Lactobacillus zeae in the patent CN108102990A disclosed above have the same effect of reducing the pH value in excrement, but the Lactobacillus zeae in the patent CN108102990A has no degradation effect of ammonia water and hydrogen sulfide.

Example 3: compounding of microbial deodorant

(1) Antagonism experiment

And performing cross-hatching on the three obtained deodorization strains on a nutrient solid culture medium by adopting a cross-hatching method, and observing the growth conditions of cross-point strains. The crossing point indicates no antagonism if it grows, and conversely indicates antagonism between strains. As a result, it was found that there was no antagonism between these three strains.

(2) Compounding of deodorizing bacterial strain

Respectively inoculating the obtained three deodorant strains into nutrient broth, YPD and MRS liquid culture media, performing shake culture to logarithmic growth phase, and compounding culture solutions of 3 strains according to the following proportion (bacillus: yeast: lactic acid bacteria): a: 0: 1; b: 1: 0: 1; c: 1: 0; d: 1: 1; e: 1: 2; f: 2: 1: 2; g: 2: 1, 7 in number, respectively adding into 1000mL plastic beakers with covers containing 200g fresh feces, and mixing well. And respectively placing 1 small beaker with the volume of 50mL containing 30mL of boric acid absorption liquid or alkaline zinc ammine salt solution in each large beaker for detecting ammonia gas or hydrogen sulfide. The results are shown in Table 4.

TABLE 4 Effect of bacterial strain combination on ammonia and hydrogen sulfide removal

Note: different combinations of pairs of NH₃And H₂S removal rate is subjected to random variance analysis, and different capitalized alphabets represent differencesDifferent significance, different lower case letters indicate extremely different significance (LSD method).

The compounding ratio of the microbial strains is the key point for improving the effect of the deodorant, and the optimal deodorization effect can be achieved only by adjusting the optimal configuration ratio to form stable microecological flora with synergistic effect. As can be seen from Table 4, the microbial deodorant compounded in different proportions has a certain deodorization effect on ammonia gas and hydrogen sulfide, when the ratio of the strains 2-2, 2-6 and 2-20 is 2: 1, the removal rates of the ammonia gas and the hydrogen sulfide reach more than 65%, and the ratio of the three strains is 1:1, the removal effects of the ammonia gas and the hydrogen sulfide are the best, and the removal rates are 73.04% and 71.79% respectively.

Example 4: preparation method of composite microbial deodorant bacterial agent

A preparation method of a composite microbial deodorant bacterial agent comprises the following steps:

(1) strain activation

Inoculating Bacillus cereus 2-2 on nutrient broth culture medium, shake culturing at 35 deg.C and 160rpm to logarithmic phase of growth to obtain Bacillus cereus 2-2 seed solution;

inoculating Pichia mansoni 2-6 on YPD culture medium, shake culturing at 32 deg.C and 160rpm until logarithmic phase of growth to obtain Pichia mansoni 2-6 seed liquid;

inoculating lactobacillus zeae 2-20 on MRS culture medium, shake culturing at 35 deg.C and 160rpm to logarithmic phase to obtain lactobacillus zeae 2-20 seed solution;

wherein, the components of the nutrient broth culture medium are as follows: 10g of peptone, 3g of beef powder, 5g of sodium chloride and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2, and the components are sterilized at 121 ℃ for 20min for later use;

the YPD medium comprises the following components: 10g of yeast extract, 20g of glucose, 20g of peptone and 1000mL of distilled water, and sterilizing at 115 ℃ for 30min for later use;

MRS culture medium: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0 mL of tween, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000mL of distilled water, 6.2-6.6 of pH, and 30min of sterilization at 115 ℃ for later use.

(2) Culture of bacterial strain

Inoculating the bacillus cereus 2-2 seed solution obtained in the step (1) into an amplification culture medium according to the inoculation amount of 10% of the volume ratio, and performing shake culture at 35 ℃ and 160rpm for 16-24h to obtain the bacillus cereus 2-2 bacterial solution, wherein the amplification culture medium comprises the following components in percentage by mass: 1% of glucose, 1% of corn starch, 0.2% of yeast extract and 1.5% of soybean meal;

inoculating the Pichia mansoni 2-6 seed solution obtained in the step (1) into an amplification culture medium according to the inoculation amount of 10% of the volume ratio, and carrying out shake cultivation at 32 ℃ and 160rpm for 24-36h to obtain the Pichia mansoni 2-6 bacterial solution, wherein the amplification culture medium comprises the following components in percentage by mass: 2% of glucose, 1% of yeast extract, 1% of peptone and KH₂PO₄ 0.15％，MgSO₄ 0.02％；

Inoculating the lactobacillus zeae 2-20 seed solution obtained in the step (1) into an amplification culture medium according to the inoculation amount of 10% of the volume ratio, and performing shake cultivation at 35 ℃ and 160rpm for 20-36h to obtain the lactobacillus zeae 2-20 bacterial solution of the waxy sample, wherein the amplification culture medium comprises the following components in percentage by mass: 5% of brown sugar, 1% of corn starch, 1% of peptone and KH₂PO₄ 0.15％，MgSO₄ 0.02％；

(3) Strain compounding

Detecting the bacterial counts of the bacillus cereus 2-2 bacterial liquid, the pichia mansoni 2-6 bacterial liquid and the lactobacillus zeae 2-20 bacterial liquid obtained in the step (2), and according to the ratio 1 of the bacterial counts of the bacillus cereus 2-2, the pichia mansoni 2-6 and the lactobacillus zeae 2-20: 1:1, mixing, and then obtaining the composite microbial deodorant bacterial agent, wherein the pH value of the mixture is 5.0-6.5; wherein the total bacteria number of the composite microbial deodorant bacterial agent is 2.5 multiplied by 10⁹one/mL.

Example 5: application of composite microbial deodorant bacterial agent

The obtained compound microbial preparation and the diluent diluted by 10 times and 100 times are added into a 1000mL plastic beaker with a cover and containing 200g of fresh excrement according to the proportion of 10 percent, and the mixture is fully and uniformly mixed. And respectively placing 1 small beaker with the volume of 50mL and filled with 30mL of boric acid absorption liquid or alkaline zinc ammine salt solution in the large beaker for detecting ammonia gas or hydrogen sulfide. The results are shown in Table 5.

TABLE 5 effect of microbial inoculum and its dilution on ammonia and hydrogen sulfide removal

Treatment of	NH₃Removal Rate (%)	H₂S removal Rate (%)
			Microbial inoculum	76.18±3.12Aa	73.80±0.73Aa
Diluting by 10 times	71.64±1.48Aa	65.93±3.66Aa
			Diluting by 100 times	52.33±5.77Bb	47.76±2.61Bb

Note: NH pair of compound microbial agent₃And H₂And (4) carrying out variance analysis on the S removal rate, wherein different capital letters represent obvious differences, and different lowercase letters represent extremely obvious differences (LSD method).

The experiment shows that the removal rates of ammonia and hydrogen sulfide of the compound microbial inoculum and 10 times of diluent thereof are both up to more than 65% after the pig manure is treated for 3 days, and the difference is obvious and unobvious. In the embodiment, the pig manure is treated for 3 days, and if the microbial inoculum is applied for a longer time, the number of bacteria in the microbial inoculum is increased, so that the microbial inoculum maintains the dominant bacterial flora, a better and more lasting effect can be produced, and the application prospect in a foul farm is good.

Example 6

The composite microbial deodorant bacterial agent was prepared according to the preparation method described in example 4, except that:

in the step (3), the ratio of the number of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 is 1:2:2, the pH value is 5.0-6.5 after mixing, and the total number of bacteria of the composite microbial deodorant microbial inoculum is 2 multiplied by 10⁹one/mL.

Detection was carried out as described in example 5, NH₃The removal rate was 69.95%, H₂The S removal rate was 67.16%.

Example 7

in the step (3), the ratio of the number of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 is 1:0.5:1, the pH value is 5.0-6.5 after mixing, and the total number of bacteria of the composite microbial deodorant microbial inoculum is 1 multiplied by 10⁹one/mL.

Detection was carried out as described in example 5, NH₃The removal rate was 70.84%, H₂The S removal rate was 65.46%.

Example 8

in the step (3), the ratio of the number of bacillus cereus 2-2, pichia mansoni 2-6 and lactobacillus zeae 2-20 is 1:1:0.5, the pH value is 5.0-6.5 after mixing, and the total number of bacteria of the composite microbial deodorant microbial inoculum is 4 multiplied by 10⁹one/mL.

Detection was carried out as described in example 5, NH₃The removal rate was 72.94%, H₂The S removal rate was 69.15%.

Claims

1. 2-6 of a strain of Pichia manshurica, which is preserved in the common microorganism center of the China general microbiological culture Collection center in 2018, 09 and 26 months, wherein the preservation addresses are as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 16529.