CN103484402A - Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung - Google Patents
Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung Download PDFInfo
- Publication number
- CN103484402A CN103484402A CN201310403129.2A CN201310403129A CN103484402A CN 103484402 A CN103484402 A CN 103484402A CN 201310403129 A CN201310403129 A CN 201310403129A CN 103484402 A CN103484402 A CN 103484402A
- Authority
- CN
- China
- Prior art keywords
- subtilis
- bacillus subtilis
- hydrogen sulfide
- buffalo
- dung
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung. The Bacillus subtilis X3 is preserved in China Center for Type Culture Collection on June 16, 2013, the serial number is CCTCC NO: M2013261, and the preservation address is Wuhan University, Wuhan, China. Soil and the like in a buffalo farm are used as strain separation sources, the Bacillus subtilis X3 is cultured at 37 DEG C for 24h and is directly inoculated into a sealed beaker filled with buffalo dung in a proportion of 20% (W/W), and the Bacillus subtilis X3 is cultured for 2-8 days at room temperature to reduce hydrogen sulfide. Compared with a control group added with an equal proportion of sterile water, the Bacillus subtilis X3 can reduce the generation of hydrogen sulfide in the buffalo dung by 19.59-52.74% and has the advantages of high efficiency, low nutrition requirements, simple operation and high safety.
Description
Technical field
The present invention relates to a strain and can reduce new bacterial strain subtilis X3 (Bacillus subtilis X3) and the application in reducing the generation of ight soil hydrogen sulfide thereof that in buffalo ight soil, hydrogen sulfide generates.
Background technology
In recent years, rapidly, the large-scale cultivation ratio constantly increases China's livestock and poultry breeding industry development, when meeting the animal products demand day by day improved and the level of improving the people's livelihood, has also brought a series of ecological problem.Livestock waste producing, process, deposit with discharge process and can produce a large amount of repugnant substances, these repugnant substance complicated components, mainly contain the chemical substances such as ammonia, sulfide, amine and voltaile fatty acid.Stench refers to that all stimulate olfactory organ cause that people are unhappy and damage bioenvironmental material.Stench is as one of 7 kinds of typical public hazards (topsoil, water pollution, stench, soil pollution, noise, vibration, land subsidence), its substance classes is various, range of influence is large, and the serious harm HUMAN HEALTH, it is many-sided to the murder by poisoning of human body: (1) harm neural system.The repugnant substance that is subject to for a long time one or more lower concentrations stimulates, and at first makes the de-mistake of sense of smell, then causes the regulatory function imbalance of pallium excitement and process of inhibition.Some repugnant substances as hydrogen sulfide not only has the foreign odor effect, also produce toxic action to neural system simultaneously; (2) harm respiratory system.When people smell foul smell, can suppress air-breathing in reflectivity ground, hinder normal respiratory function; (3) the harm recycle system.As pungent odors such as ammonia, can make blood pressure occur first descending and rising afterwards, first the slow down variation of rear quickening of pulse.Hydrogen sulfide can also hinder the conveying of oxygen, and causes anoxic in body; (4) harm Digestive tract.Often the contact repugnant substance, lose the appetite the people and feel sick, and then develop into digestive function and go down; (5) stench can make the secreting function disorder of endocrine system, and affects the Metabolic activity of body.Ammonia and aldehydes have hormesis to eyes in addition, often cause shed tears, pain, conjunctivitis, corneal edema; (6) stink also can hinder the good development of interpersonal relation, the external image in impact individual, family or a certain place.The continuous action that is subject to for a long time stench can make people's agitation, melancholy, insomnia, absent minded, hypomnesis, thereby makes the study and work Efficiency Decreasing.On human body sense organ and healthy impact, cause the generally attention of countries in the world due to stink, the research of relevant deodorization technology has become an important link in the environmental improvement engineering.
At present, the removal method for stink mainly contains: (1) chemical deodorizing method (oxidation style, absorption process, absorption method) comprises combustion method (flame combustion, catalyticcombustion); (2) physical deodorization method, as cover the method for keeping away, dilution diffusion process etc.; (3) biological odor removal method, be mainly to utilize microbial deodorant, and the physiological metabolism by microorganism is transformed tool material frowzy.For volatile organic waste gas and the foul gas of large flow, lower concentration, use the physics and chemistry method to process to exist that investment is large, complicated operation, problem that running cost is high.As far back as nineteen fifty-seven, the U.S. has just reported the patent of utilizing the soil deodorization method to process hydrogen sulfide.Biologic deodorization method because of its have applied widely, processing efficiency is high, non-secondary pollution, required equipment simple, convenient operation, running cost is low, carrying capacity of environment is low and maintenance management characteristics simply and easily, can be widely used in sewage work, disposal site, Zhong lake, park and the different places such as ditch, livestock-raising field.At beginning of the nineties late 1980s, the biological deodorizing technology is developed rapidly, and develops into gradually the main flow of deodorization technology.It is a kind ofly to utilize microorganism to decompose repugnant substance to make its odorless, innoxious treatment process, therefore also be the microbial deodorant method.Biological odor removal method divides by processing mode Biological fitler method, biological washing method, bio-trickling method etc.; By packing material, divide the soil deodorization process is arranged, pearlite wool deodorization process, compost deodorizing method, active sludge deodorization process, peat soil deodorization process and sawdust deodorization process etc.Because main composition in the foul smell from livestock and poultry cultivation or fecaluria processing field is ammonia and hydrogen sulfide, biological odor removal method also comes from just this purpose and grows up, so these methods are the most effective for the stink of eliminating ammonia and hydrogen sulfide.At present various countries to the treatment process of cow dung etc. nothing more than adopting anaerobic digestion, i.e. biogas fermentation.And really with microorganism, directly remove in cow dung at present, the research report of the foul smell flavor especially produced in buffalo ight soil is still few.
Summary of the invention
The purpose of this invention is to provide that a strain can reduce new bacterial strain subtilis X3 (Bacillus subtilis X3) that hydrogen sulfide in buffalo ight soil generates and in the application of microbiological deterioration hydrogen sulfide.
The technical scheme that the present invention solves the problems of the technologies described above is:
1. subtilis X3 (Bacillus subtilis X3), be preserved in Chinese Typical Representative culture collection center, and preservation date is on 06 16th, 2013, is numbered CCTCC NO:M2013261, the preservation address: Wuhan, China Wuhan University.
Hydrogen sulfide degradation bacteria provided by the present invention is separated near the grassland soil buffalo plant of Guangxi Zhuang Autonomous Region buffalo institute, name subtilis X3 (Bacillus subtilis X3), through morphological specificity and 16SrRNA sequence be initially identified as _, there is no at present the report that hydrogen sulfide in utilization _ reduction buffalo ight soil generates.
The form of bacterial strain: shaft-like, after 37 ℃ of solid mediums are cultivated 24 hours, bacterium colony becomes oyster white, circle, and the smooth of the edge, moistening, thicker in the middle of bacterium colony, to surrounding attenuation gradually.
Near the acquisition of this bacterial strain: sample grassland soil the buffalo plant of Guangxi Zhuang Autonomous Region buffalo institute, by tentatively, multiple sieve, acquisition has the pure growth that reduces the generation of buffalo ight soil hydrogen sulfide, this culture is carried out to form and 16SrRNA evaluation, it is accredited as to subtilis X3 (Bacillus subtilis X3).
The separation of this bacterial strain, screening: by pedotheque with 10%(W/W) ratio be inoculated in and be equipped with in fresh water cow dung beaker just, every kind of three repetitions, stir with glass rod, airtight, under room temperature, cultured continuously is 7 days, every 24h, with organoleptic method, judges deodorizing effect.If any the obviously processing of deodorizing effect, microorganism separation and purification experiment is carried out in sampling dilution immediately, adopts repeatedly plate streak to separate single bacterial strain from the sample with obvious deodorizing effect, and used medium is beef-protein medium.
The form of bacterial strain: shaft-like, after 37 ℃ of solid mediums are cultivated 24 hours, bacterium colony becomes oyster white, circle, and the smooth of the edge, moistening, thicker in the middle of bacterium colony, to surrounding attenuation gradually.
The 16SrRNA sequencing of bacterial strain: adopt machinery broken wall law by the DNA extraction of X3 bacterial strain out, after purified, select 16SrRNA sequence the censorship order-checking of primer 8f (5 '-CACGGATCCAGAGTTTGAT (C/T) is TGGCTCAG-3 ' (A/C))/1510r (5 '-GTGAAGCTTACGG (C/T) TACCTTGTTACGACTT-3 ') amplification bacterium, utilize Blast that the 16SrRNA gene order in measured sequence and GenBank database is carried out to the homology comparative analysis, in its homology and GenBank database, the homology of many subtilises is more than 99%, so by strain X 3 called after subtilis X3 (Bacillus subtilis X3), its 16SrRNA sequence following (5 ' → 3 '):
1 tctgtccacc ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa
61 actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc
121 tgatccgcga ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa
181 ctgagaacag atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc
241 attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc
301 ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag
361 atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca
421 accatgcacc acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga
481 ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg
541 cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg
601 gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc
661 atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc
721 ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta
781 cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt
841 tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc
901 gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc
961 tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa
1021 cggtacttgt tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc
1081 ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag
1141 gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat
1201 cgtcgccttg gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa
1261 gtggtagccg aagccacctt ttatgtttga accatgcggt tcaaacaacc atccggtatt
1321 agccccggtt tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc
1381 cgtccgccgc taacatcagg gagcaagctc ccatctgtcc gctcgacttg catgtattag
1441 gcacgcc
The cultivation of bacterial strain: get subtilis X3 (Bacillus subtilis X3) the bacteria suspension 2ml prepared and be inoculated in beef-protein medium, cultivate 24 hours in 37 ℃ of incubators.
The preservation of bacterial strain: after sterilizing 40% glycerine mixes, preserve in-20 ℃ of refrigerators.
Subtilis X3 of the present invention (Bacillus subtilis X3) reduces the application in hydrogen sulfide in microorganism.
50g cow dung is added in 1000ml plastics large beaker, by 20% inoculum size inoculating strain subtilis X3 nutrient solution, with the equivalent sterilized water in contrast; Put into the small beaker that fills 20ml zinc ammonium complex salt solution at a large beaker, in order to absorbing hydrogen sulphide (measuring method is shown in appendix 1).Two layers of plastic film and the sealing of one deck preservative film for large beaker, do two repetitions, under room temperature, cultivates; Every 2 days, get the burst size that small beaker detects hydrogen sulfide, continuous 8 days.Experiment showed, that the hydrogen sulfide that subtilis X3 (Bacillus subtilis X3) (being numbered CCTCC NO:M2013261) can reduce in 19.59%～52.74% buffalo ight soil effectively generates.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: for buffalo ight soil characteristics, this bacterial strain has the advantages that efficient reduction hydrogen sulfide generates, and easy and simple to handle, has a extensive future.
Embodiment
Below in conjunction with embodiment, the invention will be further described, but protection scope of the present invention is not limited to this.
Embodiment 1
The separation of subtilis X3 (Bacillus subtilis X3, be numbered CCTCC NO:M2013261).
The separation of subtilis X3 (Bacillus subtilis X3 is numbered CCTCC NO:M2013261) comprises sample primary dcreening operation and strains separation two portions:
1. contain the sample source primary dcreening operation of efficient deodorizing microorganism:
(1) respectively in the outer water drain mud in weighing cattle farm, Shui, cattle farm, fish pond water drain mud, limit, fish pond mud, meadow do cow dung 10g, putting into centrifuge tube (notes: should reserve one little section space and not fill, avoid freezing rear volume to increase expansion) ,-20 ℃ of preservations, every kind of sample is deposited 3 pipes.
(2) the fresh cow dung of weighing 20g adds culture dish.
(3) outside the weighing cattle farm, in water drain mud, Shui, cattle farm, fish pond, cow dung is done on water drain mud, limit, fish pond mud, meadow, sterilized water is inoculated into the culture dish that fresh cow dung is housed by 10% (W/W), every kind of three repetitions.Glass rod stirs.
(4) build culture dish, cultivate under room temperature.
(5) timing is observed, and every 24h, with organoleptic method, judges deodorizing effect (organoleptic method judges that the deodorizing effect standard is in Table 1), continuous 7 days.
(6) if any the obvious substratum of deodorizing effect, mark.And microorganism separation and purification experiment is carried out in sampling dilution immediately.
Table 1 organoleptic method is judged the deodorizing effect standard
Test-results is as table 2.
Table 2 different sources sample deodorizing subjective appreciation record
Table 2 result shows, adds the dry cow dung sample in meadow to removing the deodorizing effect the best in cow dung, therefore selects this sample to carry out the separation screening of deodorizing microorganism.
2. the separation of efficient deodorizing microorganism:
(1) separation of microorganism
Adopt plate streak to separate single bacterial strain from the dry cow dung sample in the interpolation meadow with obvious deodorizing effect, used medium is beef-protein medium, and specifically formula and compound method are as follows:
A. substratum forms and content:
B. compound method:
Take extractum carnis and peptone is put into beaker, add the water yield of needs, heating and melting, add sodium-chlor while stirring, with sodium hydroxide or salt acid for adjusting pH value, after adding agar to melt, in each culture dish of packing.Culture dish is put into 121 ℃ of sterilizing 20min of Autoclave after wrapping with newspaper.
(2) primary dcreening operation of deodorizing microorganism
The fresh cow dung of weighing 20g adds culture dish, in the microbial culture medium be separated in 20% ratio (W/W) inoculation (1) (cultivating through 24 hours), with glass stick, stirs.Using the equivalent sterilized water as blank.Build culture dish, cultivate under room temperature.Timing is observed, and two laboratory technicians judge deodorizing effect (table 1 organoleptic method is judged the deodorizing effect standard), continuous 5 days every 24h with organoleptic method.Record odor concentration changing conditions every day, within the 5th day, select the microorganism that more obvious deodorizing effect is arranged, mark.
Deodorizing microorganism primary dcreening operation experimental result is as table 3:
Table 3 organoleptic method is judged deodorizing effect
As can be seen from Table 3, organoleptic method judges that 3 pairs of buffalo ight soil of strain X have certain deodorizing effect.
Embodiment 2
Deodorizing microorganism subtilis X3 (Bacillus subtilis X3) reduces the mensuration that in buffalo ight soil, hydrogen sulfide generates:
(1) 50g cow dung is added in 1000ml plastics large beaker, be seeded in Primary Screening Test the microbial culture medium that deodorizing effect is arranged by 20% inoculum size, with the equivalent sterilized water in contrast;
(2) put at a large beaker small beaker that fills 20ml zinc ammonium complex salt solution, in order to absorbing hydrogen sulphide (measuring method is shown in appendix 1).Two layers of plastic film and the sealing of one deck preservative film for large beaker, do two repetitions, under room temperature, cultivates.
(3), every 2 days, get the burst size that small beaker detects ammonia and hydrogen sulfide, continuous 8 days.
Result is as follows:
Table 4 result shows, the hydrogen sulfide that bacterial strain subtilis X3 (Bacillus subtilis X3) can reduce in buffalo ight soil generates.
Table 4 subtilis X3 (Bacillus subtilis X3) reduces hydrogen sulfide burst size result
Embodiment 3
The evaluation of deodorizing microorganism subtilis X3 (Bacillus subtilis X3):
By the microorganism subtilis X3 (Bacillus subtilis X3) of the decrease hydrogen sulfide burst size that filters out, in the inoculation liquid nutrient medium, cultivate 16h in incubator, then sampling is observed under the microscope; In addition, get the 2ml sample and add the 2ml cryopreservation tube,-80 ℃ of preservations, for DNA extraction, adopt afterwards 16SrRNA sequence the censorship order-checking of primer 8f (5 '-CACGGATCCAGAGTTTGAT (C/T) is TGGCTCAG-3 ' (A/C))/1510r (5 '-GTGAAGCTTACGG (C/T) TACCTTGTTACGACTT-3 ') amplification bacterium.
Microscopic examination and sequencing result are as follows:
The microbe colony form that micro-Microscopic observation separation and Culture goes out.Colonial morphology: oyster white, circle, the smooth of the edge, moistening, thicker in the middle of bacterium colony, to surrounding attenuation gradually.Extract thallus DNA, 16SrRNA is surveyed in censorship, through with the Genbank database in existing sequence alignment, tentatively be defined as _.Be below the 16SrRNA sequence (5 '-> 3 ' of subtilis X3 (Bacillus subtilis X3)):
1 tctgtccacc ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa
61 actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc
121 tgatccgcga ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa
181 ctgagaacag atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc
241 attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc
301 ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag
361 atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca
421 accatgcacc acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga
481 ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg
541 cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg
601 gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc
661 atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc
721 ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta
781 cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt
841 tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc
901 gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc
961 tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa
1021 cggtacttgt tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc
1081 ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag
1141 gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat
1201 cgtcgccttg gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa
1261 gtggtagccg aagccacctt ttatgtttga accatgcggt tcaaacaacc atccggtatt
1321 agccccggtt tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc
1381 cgtccgccgc taacatcagg gagcaagctc ccatctgtcc gctcgacttg catgtattag
1441 gcacgcc
Appendix 1: the sulfurated hydrogen detection method is as follows:
The reagent compound method:
Zinc ammion complex salt absorption liquid: 5g zinc sulfate (ZnSO47H2O) is dissolved in 500ml water, separately gets 6g sodium hydroxide and is dissolved in 300ml water, and two kinds of solution are mixed.Add 70g ammonium sulfate under constantly stirring, after zinc hydroxide dissolves, add 50g glycerine, be diluted with water to 1L.
P-aminodimethylaniline solution: get 50ml sulfuric acid, add in 30ml water, get 12g P-aminodimethylaniline hydrochloride after cooling, be dissolved in above-mentioned solution, store in refrigerator, during use, get the 2.5ml stock solution, with 1+1 sulfuric acid, be diluted to 100ml.
Liquor ferri trichloridi: take 100g iron trichloride (FeCl
36H
2o) soluble in water, be diluted to 100ml, if precipitation is arranged, after needing to filter, use.
Mix nitrite ion: face the used time, in 1ml, to amino-xylene amine, use the ratio of liquid and 1 (0.04ml) iron trichloride to mix.(this mixed solution is wanted matching while using, if precipitation occurs, should give it up.）
40% ammonium dibasic phosphate solution: [(NH4) 2HPO4] is soluble in water for weighing 40g Secondary ammonium phosphate, and is diluted to 100ml.
0.1mol/L Sulfothiorine reference liquid: get that 26g Sulfothiorine and 0.2g anhydrous sodium carbonate are dissolved in that 1L newly boils and cooling water in, shake up, store in brown bottle, place and demarcate afterwards in three days.If muddiness should be filtered.
0.01mol/L Sulfothiorine reference liquid: go the calibrated 0.1mol/L hypo solution of 100ml in the brown volumetric flask of 1L, with newly boiling and cooling water is diluted to scale and shakes up.
0.1mol/L iodine reference liquid: weighing 40g potassiumiodide, be dissolved in 25mL water, then weighing 12.7g iodine, be dissolved in liquor kalii iodide, and dilute with water 1L.
0.01mol/L iodine solution: accurately the 0.1mol/L iodine solution of pipette 100mL is in the brown volumetric flask of 1L, and another weighing 18g potassiumiodide is dissolved in a small amount of water, moves into volumetric flask, is diluted with water to scale.
0.5% starch solution: weighing 0.5g Zulkovsky starch, after adding 5mL water furnishing pasty state, then add in 100mL boiling water, and boil 2-3min, transparent to solution, cooling, face and join existing use.
The 1:1 hydrochloric acid soln: the 50mL concentrated hydrochloric acid mixes with the 50mL water.
Sodium sulphite stock solution: get sodium sulfide crystal (Na
2s9H
2o), by a small amount of water clean surface, with filter paper, blot.Weighing 0.71g sodium sulfide crystal, be dissolved in and newly boil in cooling water, and redilution is to IL.By iodimetry,iodometry, demarcated.
Sodium sulphite reference liquid: after demarcation, immediately according to the concentration of sodium sulphite stock solution, calculate the needed stock solution volume of preparation 500ml5 μ g/mL sodium sulphite reference liquid Vc, add Vc sodium sulphite stock solution in the 500ml brown bottle, with newly boiling also cooling water, be diluted to scale, shake up.(should do immediately typical curve, standardized solution must newly be joined at every turn, the existing demarcation, the existing use.）
Scaling method:
Accurately the standardized solution of pipette 20ml0.01N iodine is in the 250ml iodine flask.Add 90ml water, add the 1ml1+1 hydrochloric acid soln, accurately add the 10.00ml sodium sulfide solution, mix, be placed on dark place 3min.With the 0.01N sodium thiosulfate standard solution, be titrated to light yellow again, add 0.5% starch fluid of the new preparation of 1ml to be blue, with a small amount of water washing bottle inwall, continuing to be titrated to blueness just disappears (owing to there being sulphur to generate again, make solution be micro-muddy look, now will pay special attention to the titration end point color change).Record the volume of sodium thiosulfate standard solution used, separately get 10ml water and do blank titration, its titration step is identical, records the volume of blank titration sodium thiosulfate standard solution used simultaneously.Sample titration and blank titration respectively repeat 2 times.The volumetric errors of twice titration Sulfothiorine used is no more than 0.05ml.
The concentration of hydrogen sulfide calculation formula:
C=（V2-V1）×N×17/10
In formula, C: the concentration of hydrogen sulfide, mg/mL
V2: the volume of sample titration Sulfothiorine used, mL
V1: the volume of blank titration Sulfothiorine used, mL
N: sodium thiosulfate standard solution (mg/mL)
17: concentration of hydrogen sulfide (mg/mL).
Sequence table
Claims (5)
1. subtilis X3 (Bacillus subtilis X3), be preserved in Chinese Typical Representative culture collection center, and preservation date is on 06 16th, 2013, is numbered CCTCC NO:M2013261, the preservation address: Wuhan, China Wuhan University.
Subtilis X3 as claimed in claim 1 (Bacillus subtilis X3) _, it is characterized in that, described subtilis X3 (Bacillus subtilis X3) is characterized as: shaft-like, after 37 ℃ of solid mediums are cultivated 24 hours, bacterium colony becomes oyster white, circle, the smooth of the edge, moistening, thicker in the middle of bacterium colony, to surrounding attenuation gradually.
3. subtilis X3 as claimed in claim 1 (Bacillus subtilis X3) is characterized in that the 16SrRNA sequence following (5 ' → 3 ') of subtilis X3 (Bacillus subtilis X3):
1 tctgtccacc ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa
61 actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc
121 tgatccgcga ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa
181 ctgagaacag atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc
241 attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc
301 ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag
361 atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca
421 accatgcacc acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga
481 ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg
541 cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg
601 gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc
661 atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc
721 ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta
781 cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt
841 tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc
901 gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc
961 tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa
1021 cggtacttgt tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc
1081 ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag
1141 gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat
1201 cgtcgccttg gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa
1261 gtggtagccg aagccacctt ttatgtttga accatgcggt tcaaacaacc atccggtatt
1321 agccccggtt tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc
1381 cgtccgccgc taacatcagg gagcaagctc ccatctgtcc gctcgacttg catgtattag
1441 gcacgcc 。
4. subtilis X3 as claimed in claim 1 (Bacillus subtilis X3) application of hydrogen sulfide in microbiological deterioration ight soil.
5. the application of subtilis X3 as claimed in claim 1 (Bacillus subtilis X3) in reducing the generation of buffalo ight soil hydrogen sulfide, it is characterized in that: subtilis X3 (Bacillus subtilis X3) is carried out to enlarged culturing, then be inoculated in ight soil, the hydrogen sulfide in ight soil is reduced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310403129.2A CN103484402A (en) | 2013-09-06 | 2013-09-06 | Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310403129.2A CN103484402A (en) | 2013-09-06 | 2013-09-06 | Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103484402A true CN103484402A (en) | 2014-01-01 |
Family
ID=49824998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310403129.2A Pending CN103484402A (en) | 2013-09-06 | 2013-09-06 | Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103484402A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107384817A (en) * | 2016-03-28 | 2017-11-24 | 财团法人酦酵微生物产业振兴院 | With enzymatic activity, the bacillus subtilis strain for reducing the effect of ammonia and hydrogen sulfide and application thereof |
| CN111733118A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus PL-2 and application thereof in aquaculture |
| CN111849828A (en) * | 2020-08-04 | 2020-10-30 | 榆林沙漠王生物科技有限公司 | Compound microbial inoculum for decomposed compost of livestock and poultry manure as well as preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996055A (en) * | 1989-06-05 | 1991-02-26 | Kurasawa Optical Industry Co., Ltd. | Deodorized bacteria composition |
| JP2005185658A (en) * | 2003-12-26 | 2005-07-14 | Kazunari Hashimoto | Deodorizing agent, deodorizer using it, and deodorizing method |
| CN101638626A (en) * | 2009-09-10 | 2010-02-03 | 惠飞 | Bacillus subtilis and application thereof in biological deodorant |
| CN102851231A (en) * | 2012-05-10 | 2013-01-02 | 海南芳绿源科技开发有限公司 | Functional strain, and microbial agent and application thereof |
-
2013
- 2013-09-06 CN CN201310403129.2A patent/CN103484402A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996055A (en) * | 1989-06-05 | 1991-02-26 | Kurasawa Optical Industry Co., Ltd. | Deodorized bacteria composition |
| JP2005185658A (en) * | 2003-12-26 | 2005-07-14 | Kazunari Hashimoto | Deodorizing agent, deodorizer using it, and deodorizing method |
| CN101638626A (en) * | 2009-09-10 | 2010-02-03 | 惠飞 | Bacillus subtilis and application thereof in biological deodorant |
| CN102851231A (en) * | 2012-05-10 | 2013-01-02 | 海南芳绿源科技开发有限公司 | Functional strain, and microbial agent and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 樊杰 等: "一株硝化脱氮除臭菌的筛选鉴定及其多途径氮代谢功能的研究", 《环境科学学报》, vol. 30, no. 4, 30 April 2010 (2010-04-30), pages 722 - 728 * |
| 郭军蕊 等: "畜禽养殖场除臭技术研究进展", 《动物营养学报》, vol. 25, no. 8, 24 May 2013 (2013-05-24), pages 1708 - 1714 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107384817A (en) * | 2016-03-28 | 2017-11-24 | 财团法人酦酵微生物产业振兴院 | With enzymatic activity, the bacillus subtilis strain for reducing the effect of ammonia and hydrogen sulfide and application thereof |
| CN107384817B (en) * | 2016-03-28 | 2021-04-27 | 财团法人酦酵微生物产业振兴院 | Bacillus subtilis strain with enzyme activity and effects of reducing ammonia and hydrogen sulfide and application thereof |
| CN111849828A (en) * | 2020-08-04 | 2020-10-30 | 榆林沙漠王生物科技有限公司 | Compound microbial inoculum for decomposed compost of livestock and poultry manure as well as preparation method and application thereof |
| CN111733118A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus PL-2 and application thereof in aquaculture |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103980010B (en) | Livestock and poultry manure decomposing inoculant, use method thereof and biological organic fertilizer prepared by using livestock and poultry manure decomposing inoculant | |
| CN108048344B (en) | Two plants of deodorization bacterial strains and its application in preparation composite biological deodorant | |
| CN103966149B (en) | A kind of house refuse and sludge decomposing microbial inoculum and its application and made decomposed matrix | |
| CN103484370B (en) | A kind of method utilizing farm animal excrement waste water to cultivate spirulina | |
| CN105272472A (en) | Biogas slurry formula fertilizer through fermentation of primary pig manure and urine and preparation method thereof | |
| CN102247611A (en) | Microorganism deodorant and preparation method thereof | |
| CN104974962B (en) | One plant of acinetobacter phosphorus decomposing growth-promoting bacterium Y40 and its application | |
| CN104593303B (en) | A kind of liquid complex micro organism fungicide and its production method | |
| CN104628429B (en) | A kind of preparation method of photosynthetic bacterial fertilizer | |
| CN107058153A (en) | A kind of biological deodorizing composite bacteria agent and its preparation method and application | |
| CN103937695A (en) | Composite biological bacterial agent for treating livestock and poultry breeding wastewater and manufacturing method thereof | |
| CN104195084A (en) | Diethylstilbestrol degradation strain and application thereof in sewage and animal manure treatment | |
| CN104726378A (en) | Method for improving protective enzyme activities of salt-stressed turfgrass by adopting enhanced salt-tolerant microbial agent | |
| CN105112319B (en) | One pseudomonas category phosphate-solubilizing bacteria Y2 and its biological organic fertilizer and the application of preparation | |
| KR20110040394A (en) | Removal of livestock's fetor by using microbes and complex mineral group solution | |
| CN103484402A (en) | Bacillus subtilis X3 and application thereof for reducing generation of hydrogen sulfide in buffalo dung | |
| CN107828657A (en) | A kind of preparation method of microbial bacterial agent for compost fermentation deodorization | |
| CN110699419A (en) | Method for researching bacterial function based on duckweed | |
| CN105296355A (en) | Environmental microorganism bacterium extraction and propagation compositions and using methods thereof | |
| CN103740613B (en) | A kind of preparation method of biological active bacterium agent | |
| CN103961735B (en) | A kind of microbial deodorant and preparation method thereof | |
| CN108531412A (en) | A kind of preparation method of ECMD Efficient Rings control microbial deodorant | |
| CN105219680A (en) | A kind of preparation method of photosynthetic bacterium microbial inoculum and application thereof | |
| CN105132332A (en) | Acetobacter gluconicum and application of acetobacter gluconicum as plant growth-promoting rhizobacteria | |
| CN108102943A (en) | A kind of efficient denitrification microorganism and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C05 | Deemed withdrawal (patent law before 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140101 |