CN108977399A - One plant of Bacillus foecalis alkaligenes and its application - Google Patents
One plant of Bacillus foecalis alkaligenes and its application Download PDFInfo
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Abstract
The invention belongs to microbe application fields, and in particular to a kind of Bacillus foecalis alkaligenes and its application.The specific technical proposal is: one plant of Bacillus foecalis alkaligenes, on June 11st, 2018, in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, strain name: Bacillus foecalis alkaligenes DFN2, deposit number were CGMCC No.15926.The bacterium has splendid degradation effect to common odorant such as ammonia, hydrogen sulfide etc., can reach 100% to the most degradation rate of ammonia and hydrogen sulfide；99.5% is also up to the ammonia nitrogen substances degradation rate in high ammonia-nitrogen wastewater.Bacillus foecalis alkaligenes provided by the invention can also carry out biological desulphurization processing to thiophene-type sulfide, and desulfurization effect is much better than general hydrodesulfurization effect.
Description
Technical field
The invention belongs to microbe application fields, and in particular to one plant of Bacillus foecalis alkaligenes and its application.
Background technique
As diesel oil demand increasingly increases, China is continuously increased crude conversion ability, and diesel oil sulfur content is consequently increased.Mesh
The preceding main method using hydrodesulfurization also has part naphthalene series aromatics to add hydrogen raw although the technique can increase desulfuration efficiency
At naphthane, make oil product unstability, increases desulfurization expense；Meanwhile the sulfur method reaction condition requires relatively high, cost
Also relatively high.
Start to be dedicated to studying biological desulfurizing technology currently, having many countries.Biological desulfurizing technology can be in room temperature, normal pressure
Lower progress, desulphurization cost is low, and can remove the dibenzothiophenes substance for being not easy to deviate from hydrogenation process.Biological desulphurization skill
Though art has many benefits, the presently found bacterial strain that can destroy benzothiophene substance is considerably less, just also in research and development
Stage beginning.
Meanwhile as people's living standard is continuously improved, fowl and animal excrement and house refuse also increase day by day increase, the stench of generation
Pollutant can generate a large amount of ammonias and hydrogen sulfide in landfill and composting process, not only cause sternly to the body of people with increase
It endangers, also pollutes the environment, polluted underground water source etc. again.
If a kind of bacterium can be provided, thiophene-type sulfide and odorant can be handled simultaneously, there will be pole
Big realistic meaning.
Summary of the invention
The object of the present invention is to provide one plant of Bacillus foecalis alkaligenes and its applications.
For achieving the above object, the technical scheme adopted by the invention is that: one plant of Bacillus foecalis alkaligenes, in 2018 6
The moon 11, in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, strain name: Bacillus foecalis alkaligenes
DFN2, deposit number are CGMCC No.15926.
Correspondingly, one plant of Bacillus foecalis alkaligenes, 16Sr DNA sequence dna is as shown in SEQ ID NO 1.
Correspondingly, application of the Bacillus foecalis alkaligenes in processing ammonia, hydrogen sulfide and thiophene-type sulfide.
Preferably, the condition of the Bacillus foecalis alkaligenes processing ammonia, hydrogen sulfide are as follows: temperature is 30~35 DEG C.
Correspondingly, application of the Bacillus foecalis alkaligenes in processing ammonia nitrogen waste water.
Preferably, the condition of the Bacillus foecalis alkaligenes processing ammonia nitrogen waste water are as follows: pH=5~10, temperature are 20~40 DEG C.
Preferably, the condition of the Bacillus foecalis alkaligenes processing ammonia nitrogen waste water are as follows: pH=7, temperature are 30 DEG C.
The invention has the following advantages:
1, the present invention provides a kind of Bacillus foecalis alkaligenes, which has common odorant (ammonia, hydrogen sulfide) splendid
Degradation effect, 100% can reach to the most degradation rate (removal rate) of ammonia and hydrogen sulfide；To the ammonia in high ammonia-nitrogen wastewater
Nitrogen material degradation rate is also up to 99.5%.
2, Bacillus foecalis alkaligenes provided by the invention can also carry out biological desulphurization processing, desulfurization effect to thiophene-type sulfide
It is much better than general hydrodesulfurization effect, 100% is equal to the treatment effeciency of part thiophene-type sulfide.
Detailed description of the invention
Fig. 1 is the phyletic evolution tree graph of Bacillus foecalis alkaligenes；
Fig. 2 is colonial morphology schematic diagram of the Bacillus foecalis alkaligenes in plating medium；
Fig. 3 is Bacillus foecalis alkaligenes electron-microscope scanning figure；
Fig. 4 is Bacillus foecalis alkaligenes Gram's staining schematic diagram；
Fig. 5 is influence schematic diagram of the C/N to Bacillus foecalis alkaligenes degradation of ammonia nitrogen；
Fig. 6 is influence schematic diagram of the pH to Bacillus foecalis alkaligenes degradation of ammonia nitrogen；
Fig. 7 is influence schematic diagram of the revolving speed to Bacillus foecalis alkaligenes degradation of ammonia nitrogen；
Fig. 8 is influence schematic diagram of the temperature to Bacillus foecalis alkaligenes degradation of ammonia nitrogen；
Fig. 9 is the degradation effect curve graph of Bacillus foecalis alkaligenes degradation of ammonia nitrogen；
Figure 10 is Bacillus foecalis alkaligenes degradation high ammonia-nitrogen wastewater effect diagram；
Specific embodiment
The culture medium that full text is related to is as follows:
1, Vickers salting liquid prepares (g/L): K2HPO45.0, MgSO4·7H2O 2.5, NaCl 2.5, FeSO4·7H2O
0.05, MnSO40.05, pH=7.0.
2, enriched medium is (g/L): ammonium sulfate 2.0；Sodium succinate 13.85；Sodium acetate 5.0；Glucose 5.0；Vickers
Salting liquid 50mL, pH=7, add water to 1L.
3, isolation medium (g/L): (NH4)2SO40.47, sodium succinate 5.62, Vickers salting liquid 50mL, pH=7.0.
4, separate solid medium: it is isolation medium volume 2.0% that volume is added on the basis of isolation medium
Agar, 121 DEG C, the processing of 30min high-temperature sterilization.For nitrification bacteria isolate and purify and deamination characteristic research.
5, beef-protein medium: beef extract 5g, peptone 10g, NaCl 5g add water to 1L, pH=7,121 DEG C,
The processing of 30min high-temperature sterilization.
6, beef extract-peptone solid medium: it is beef extract that volume is added on the basis of beef-protein medium
The agar of peptone solid medium volume 2.0%.Morphologic observation, preservation and activation for bacterial strain.
7, liquid expands fermentation medium: beef extract 5g, glucose 10g, peptone 5g, change sodium 5g, adds water to 1000ml,
PH 7.0~7.4.
Embodiment one: the acquisition and identification of bacterial strain
1, it acquires: Sichuan secondary sedimentation tank of sewage treatment work sludge being taken to mix with compost sample.By the activated sludge of acquisition with
Compost sample mixing is inoculated in enriched medium, 30 DEG C, aerobic condition, by 10% that volume is enriched medium with enrichment
Culture medium is tamed, and per generation tames 5 days, and it is 20 days total to tame for 4 generations altogether, gradient dilution is then carried out, from 10-2、10-3、10-4、
10-5、10-6、10-7、10-80.25mL is drawn under dilution, is coated in isolation medium plate, 30 DEG C of constant temperature incubations are repeatedly divided
Several plants of bacterial strains are finally obtained from rear.
The compost sample refer specifically to by common crops by-product (such as stalk, rice husk) directly stack 150 days and with
On.
2, Molecular Identification
Using Solarbio bacterial genomes DNA extraction kit, the genomic DNA of bacterial strain is extracted, as template, benefit
With 16SrDNA gene universal primer, PCR amplification is carried out.Expand the primer of 16SrDNA:
F-primerF27:5 '-AGA GTT TGA TCC TGG CTC AG-3 '；
R-primerR1492:5 '-GGC TAC CTT GTT ACG ACT T-3 '.
PCR reaction system are as follows: 2 × mix:12.5 μ L, F-primerF27:1.0 μ L, R-primerR1942:1.0 μ L, DNA
Genome: 1.0 μ L μ L, dd H2O:9.5 μ L.
PCR reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 100s, 29
A circulation, 72 DEG C of final extension 5min, 4 DEG C of preservations.The PCR reaction product of 3 μ L is taken to carry out 1% agarose gel electrophoresis reality
It tests.
Obtained 16SrDNA sequence product is sequenced by Chengdu Qing Ke biotechnology Services Co., Ltd.The sequence measured
Column log in the website NCBI, are compared by Blast program and Genbank amplifying nucleic acid data.Sequencing sequence such as SEQ ID
Shown in NO 1.Using MEGA6 Software on Drawing bacterial strain evo-devo tree, as shown in Figure 1.
It can be seen that the bacterial strain and Alcaligenes faecalis subsp.phenolicus from sequencing result and Fig. 1
The genetic distance of DSM 16503 (Alcaligenes faecalis DSM 16503) is nearest, determines therefrom that the bacterial strain is Bacillus foecalis alkaligenes
(Alcaligenes faecalis).The bacterial strain is on June 11st, 2018, in China Committee for Culture Collection of Microorganisms
Common micro-organisms center (China General Microbiological Culture Collection Center) preservation,
Preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Strain name: Bacillus foecalis alkaligenes (Alcaligenes
Faecalis) DFN2, deposit number are CGMCC No.15926.
3, physiological and biochemical property is identified
The plate culture colonial morphology of bacterial strain is observed, as shown in Figure 2: bacteria colony white protrusion, neat in edge is smooth, and surface is wet
Profit.
Observe thalli morphology under scanning electron microscope, as shown in figure 3, thallus be it is rod-shaped, size is about 0.4 × (0.9~1.5)
μm.Gram's staining is carried out to bacterium solution using Solarbio gram staining liquid, it is blue for leather to identify bacterial strain DNF2 by dyeing
Family name's negative bacterium, as shown in Figure 4.The bacterium can be 30~35 DEG C in 15~45 DEG C of range growths, optimum growth temperature；Can pH 5~
Growth, the most suitable growth pH 7.0~8.0 in 10 ranges.
Embodiment two: the condition of strains for degrading ammonia nitrogen substances is preferred
Inventor has found that the bacterial strain has the performance of splendid degradation of ammonia nitrogen in an experiment, can be applied to ammonia nitrogen object
In the processing of matter.In the present embodiment, used medium all carries out following corresponding adjustment on the basis of isolation medium.
The bacterial strain isolated and purified is inoculated into beef-protein medium by inoculum concentration for an oese, 35 DEG C
Culture is for 24 hours to get seed liquor.
1, influence of the C/N to ammonia nitrogen degradation
C/N refers to C, N mass ratio in culture medium.
Seed liquor is inoculated into the culture that C/N ratio is respectively 2,5,10,15,20 by volume for culture volume 1% respectively
In base, degradation rate and OD of the measurement bacterial strain to ammonia nitrogen600。
As shown in figure 5, bacterial strain can be grown under conditions of C/N ratio is respectively 2,5,10,15,20.When C/N is 2,5,
Ammonia nitrogen degradation rate is 28.9%, 61.2%, OD600Value is 0.36,0.68.When C/N ratio is 10, ammonia nitrogen degradation rate reaches maximum, is
100%, OD600Value is 1.05.With the increase of C/N ratio, ammonia nitrogen degradation rate is 100%, and cell density slightly rises.Thus
It is found that the best C/N of bacterial strain DNF2 degradation of ammonia nitrogen is 10.
2, influence of the pH value to ammonia nitrogen degradation
Seed liquor is inoculated into the culture that pH value is respectively 4,5,6,7,8,9,10 by volume for culture volume 1% respectively
In base, degradation rate and OD of the measurement bacterial strain to ammonia nitrogen600。
As shown in fig. 6, bacterial strain has wider pH value domain, can be grown in 5~10 ranges.Bacterial strain under strongly acidic conditions,
The ammonia nitrogen degradation rate of bacterial strain is 0, OD600Value is 0.02, and thallus is not grown substantially.Under the conditions of pH is 5,6,7,8,9, bacterial strain
Ammonia nitrogen degradation rate is 100%, the OD of bacterial strain600Value illustrates that strain growth is preferable 1.1 or more.Under conditions of highly basic
(pH=10), the ammonia nitrogen degradation rate of bacterial strain is 42%, OD600It is 0.68.So selecting pH=7 for best degradation condition.
3, influence of the revolving speed to ammonia nitrogen degradation
Seed liquor is inoculated into the culture medium that revolving speed is respectively 80,120,160,200r/min by volume for culture medium 1%
In, degradation rate and OD of the measurement bacterial strain to ammonia nitrogen600。
As shown in fig. 7, under the conditions of 80r/min, 120r/min, bacterial strain is respectively 48.6% to the degradation rate of ammonia nitrogen,
79.7%, OD600Value is 0.56,0.88.When revolving speed is 160r/min, bacterial strain is 100%, OD to the degradation rate of ammonia nitrogen600Value
It is 1.07.When revolving speed reaches 200r/min, bacterial strain is 87.8%, OD to the degradation rate of ammonia nitrogen600Value is 1.01.To sum up, when turn
Speed is 160r/min, ammonia nitrogen degradation rate and cell density OD600Value reaches maximum, it is known that the best degradation speed conditions of bacterial strain
For 160r/min.
4, influence of the temperature to ammonia nitrogen degradation
By seed liquor by volume be culture volume 1% be inoculated into respectively temperature condition be respectively 10 DEG C, 20 DEG C, 30 DEG C,
40 DEG C, in 50 DEG C of culture medium, degradation rate and OD of the measurement bacterial strain to ammonia nitrogen600。
As shown in figure 8, (10 DEG C, 50 DEG C) bacterial strain is not grown under low temperature and hot conditions.When temperature is 20 DEG C, bacterial strain
Degradation rate to ammonia nitrogen is 11.2%, OD600It is 0.1.When temperature is 30 DEG C, bacterial strain is 96.3% to the degradation rate of ammonia nitrogen,
OD600Value is 1.16.When temperature is 40 DEG C, bacterial strain falls to 93.6%, OD to the degradation rate of ammonia nitrogen later600Value is 1.1.Thus
It is found that the optimum temperature of bacterial strain DNF2 degradation of ammonia nitrogen is 30 DEG C.
Under the conditions of best C/N, pH of above-mentioned screening, revolving speed, temperature, bacterial strain DNF2 is inoculated into ammonia nitrogen removal culture medium
In.As shown in figure 9, bacterial strain DNF2 has preferable ammonia nitrogen removal efficiency, it can be in 15h by the ammonia nitrogen degradation of 96mg/L to 0mg/L.It is flat
Equal degradation rate is 6.4mg/Lh, and most degradation rate is 13.3mg/Lh.
Embodiment three: bacterial strain degrades to high ammonia-nitrogen wastewater
1, seed liquor prepared by embodiment two is seeded to liquid by 1% that inoculum concentration is liquid expansion fermentation medium volume
Body expands in fermentation medium, and 35 DEG C, shaken cultivation for 24 hours, obtains inoculation liquid under the conditions of 160rmp.
2, by the inoculation liquid 5ml be inoculated in equipped with 500ml biogas slurry high ammonia-nitrogen wastewater (initial ammonia nitrogen concentration be 416.2mg/
L in 1L beaker) (inoculum concentration 1.0%), while equal amount of distilled water is added as a control group.Three repetitions of every group of setting are protected
Holding temperature is 30 DEG C, and every 6h measures ammonia nitrogen concentration, as a result as shown in Figure 5.
3, the result shows that: initial ammonia nitrogen concentration be 416.2mg/L under the conditions of, bacterial strain can arrive ammonia nitrogen degradation in 84h
8.53mg/L, degradation rate 98.6%, in 96h, ammonia nitrogen concentration 2.4mg/L, degradation efficiency 99.5%.
Example IV: bacterial strain is in deodorization reactor to NH3And H2The processing of S
1,30~35 DEG C, inoculation liquid prepared by embodiment three is seeded in deodorization reactor by 1% inoculum concentration, not
Under same concentration, different load, NH is detected3And H2The removal situation of S.The results are shown in Table 1.
1 bacterial strain of table is to NH3And H2The removal situation of S is shown
2, as shown in Table 1, after Bacillus foecalis alkaligenes DNF2 is inoculated into bio-trickling filter, bacterial strain is to foul smell hydrogen sulfide and ammonia
Gas can be removed preferably, and efficiency is 94% or more.
Embodiment five: application of the bacterial strain in processing thiophene-type sulfide
Inventor has found that the bacterial strain can also be used in the desulfurization process of thiophene-type sulfide under study for action, this may be because
The enzyme that the bacterial strain generates can convert water-soluble sulfide for thiophene-type sulfide, to realize the de- of thiophene-type sulfide
Sulphur.Specific effect is presented below:
1, using commercially available ordinary diesel oil as raw material, the distribution situation of Major Sulfides therein is measured, the results are shown in Table 2.
Sulfide distribution situation table in diesel oil before table 2 is handled
2, inoculation liquid prepared by embodiment three is seeded to diesel oil culture medium by the inoculum concentration that volume is culture volume 3%
In, the diesel oil culture medium is on the basis of beef-protein medium, and addition volume is beef-protein medium
The diesel oil of volume 10% cultivates 48h, observes the growing state of bacterial strain, and detect the content of all kinds of sulfide in culture medium,
The results are shown in Table 3.Wherein, "/" expression is not detected.
After the processing of table 3 in culture medium each sulfide distribution situation
| Sulfur-containing compound | It is distributed (mg/L) |
| 4-MDBT | 4.6 |
| 4- ethyl dibenzothiophenes | 3.9 |
| 4.6- dimethyl Dibenzothiophene | 0.1 |
| 2.4- dimethyl Dibenzothiophene | / |
| 2.8- dimethyl Dibenzothiophene | / |
| 1,2- dimethyl Dibenzothiophene | 6.5 |
| 2,4,6- trimethyl dibenzothiophenes | 2.4 |
Strain growth is in good condition.From table 3 it can be seen that all kinds of thiophene-type sulfides in diesel oil, especially 2.4- bis-
Methyldibenzothiophene and 2.8- dimethyl Dibenzothiophene, degradation effect is obvious, and degradation rate is equivalent to up to 100%.
Sequence table
<110>Shaanxi Polytechnics
Chengdu Inst. of Biology, Chinese Academy of Sciences
<120>one plants of Bacillus foecalis alkaligenes and its applications
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 1368
<212> DNA
<213>Bacillus foecalis alkaligenes (Alcaligenes faecalis)
<400> 1
ttgctctctt ggcggcgagt ggcggacggg tgagtaatat atcggaacgt gcccagtagc 60
gggggataac tactcgaaag agtggctaat accgcatacg ccctacgggg gaaagggggg 120
gatcgcaaga cctctcacta ttggagcggc cgatatcgga ttagctagtt ggtggggtaa 180
aggctcacca aggcaacgat ccgtagctgg tttgagagga cgaccagcca cactgggact 240
gagacacggc ccagactcct acgggaggca gcagtgggga attttggaca atgggggaaa 300
ccctgatcca gccatcccgc gtgtatgatg aaggccttcg ggttgtaaag tacttttggc 360
agagaagaaa aggtatcccc taatacggga tactgctgac ggtatctgca gaataagcac 420
cggctaacta cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta atcggaatta 480
ctgggcgtaa agcgtgtgta ggcggttcgg aaagaaagat gtgaaatccc agggctcaac 540
cttggaactg catttttaac tgccgagcta gagtatgtca gaggggggta gaattccacg 600
tgtagcagtg aaatgcgtag atatgtggag gaataccgat ggcgaaggca gccccctggg 660
ataatactga cgctcagaca cgaaagcgtg gggagcaaac aggattagat accctggtag 720
tccacgccct aaacgatgtc aactagctgt tggggccgtt aggccttagt agcgcagcta 780
acgcgtgaag ttgaccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac 840
ggggacccgc acaagcggtg gatgatgtgg attaattcga tgcaacgcga aaaaccttac 900
ctacccttga catgtctgga aagccgaaga gatttggccg tgctcgcaag agaaccggaa 960
cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020
cgagcgcaac ccttgtcatt agttgctacg caagagcact ctaatgagac tgccggtgac 1080
aaaccggagg aaggtgggga tgacgtcaag tcctcatggc ccttatgggt agggcttcac 1140
acgtcataca atggtcggga cagagggtcg ccaacccgcg agggggagcc aatctcagaa 1200
acccgatcgt agtccggatc gcagtctgca actcgactgc gtgaagtcgg aatcgctagt 1260
aatcgcggat cagaatgtcg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca 1320
caccatggga gtgggtttca ccagaagtag gtagcctaac cgtaagga 1368
Claims (9)
1. one plant of Bacillus foecalis alkaligenes, it is characterised in that: on June 11st, 2018, in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, strain name: Bacillus foecalis alkaligenes DFN2, deposit number are CGMCC No.15926.
2. one plant of Bacillus foecalis alkaligenes, it is characterised in that: 16Sr DNA sequence dna is as shown in SEQ ID NO 1.
3. Bacillus foecalis alkaligenes as claimed in claim 1 or 2, it is characterised in that: the preparation method of the Bacillus foecalis alkaligenes are as follows: take activity
Sludge is mixed with compost sample, by 5~10% that volume is enriched medium, is inoculated in enriched medium, 30 DEG C, aerobic item
Part 15~20d of culture, is diluted to 10-8Dilution, absorption≤0.5mL is coated in isolation medium plate, the training of 30 DEG C of constant temperature
Support to get；
The compost sample refer to by crop by-products stack 150 days or more to get；
The formula of the enriched medium are as follows: ammonium sulfate 2.0g；Sodium succinate 13.85g；Sodium acetate 5.0g；Glucose 5.0g；Dimension
Family name salting liquid 50mL, adds water to 1L to obtain the final product；
The formula of the isolation medium are as follows: (NH4)2SO40.47g, sodium succinate 5.62g, Vickers salting liquid 50mL；
The Vickers salting liquid are as follows: contain K in every liter of water2HPO45.0g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·
7H2O 0.05g, MnSO4 0.05g。
4. application of the Bacillus foecalis alkaligenes as claimed in claim 1 or 2 in processing odorant.
5. application of the Bacillus foecalis alkaligenes in processing odorant according to claim 4, it is characterised in that: Bacillus foecalis alkaligenes
Application in processing ammonia, hydrogen sulfide and thiophene-type sulfide.
6. application of the Bacillus foecalis alkaligenes in processing foul gas according to claim 5, it is characterised in that: the excrement produces alkali
Bacillus handles the condition of ammonia, hydrogen sulfide are as follows: temperature is 30~35 DEG C.
7. application of the Bacillus foecalis alkaligenes as claimed in claim 1 or 2 in processing ammonia nitrogen waste water.
8. application of the Bacillus foecalis alkaligenes in processing ammonia nitrogen waste water according to claim 7, it is characterised in that: the excrement produces alkali
The condition of bacillus processing ammonia nitrogen waste water are as follows: pH=5~10, temperature are 20~40 DEG C.
9. application of the Bacillus foecalis alkaligenes in processing ammonia nitrogen waste water according to claim 8, it is characterised in that: the excrement produces alkali
The condition of bacillus processing ammonia nitrogen waste water are as follows: pH=7, temperature are 30 DEG C.
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