CN105950495B - One plant of Methylotrophic bacillus and its application in livestock breeding wastewater processing - Google Patents
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Abstract
The invention discloses a kind of bacillus HB13000 and its applications in livestock breeding wastewater processing, belong to microorganisms technical field.The bacillus is Methylotrophic bacillus (Bacillus methylotrophicus) HB13000, is located away from Hainan Province Ba Menwan mangrove bed mud sample.The bacterial strain is preserved in Chinese microorganism strain preservation management common micro-organisms center on January 18th, 2016, and deposit number is CGMCC No.12064.The bacillus HB13000 has protein degradation matter, fat, starch and cellulolytic activity, can be effectively reduced the COD value of livestock breeding wastewater.Bacillus HB13000 and its fermentation liquid can be used as a kind of new microbial resources for livestock breeding wastewater processing, have good application and development prospect in terms of wastewater treatment.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Methylotrophic bacillus HB13000 and its
Application in livestock breeding wastewater processing.
Background technique
As agricultural restructuring and agriculture industrialization promote, China's scale, intensive livestock and poultry breeding industry obtain fast
Exhibition is hailed, and thus bring problem of environmental pollution is also got worse, especially the waste water that generates of livestock and poultry farm, discharge amount
Greatly, organic pollutant concentration is high, also containing pollutants such as heavy metal, residual animal medicine and pathogen, has resulted in water source, soil and ring
The serious pollution of border air.It is reported that Pollution from livestock and poultry has become the third-largest pollution after industrial pollution, domestic pollution
One of the main reason for source is China's rural pollution.Processing livestock breeding wastewater pollution at present mainly has returning to the field method, handles naturally
Method and three kinds of biological treatment.Biological treatment is divided into Anaerobic Treatment method, Aerobic Process for Treatment method and anaerobic-aerobic combined method again,
It is that the pollutant in waste water is decomposed and converted using microorganism, is a kind of to eliminate pollution and harm to environment
Handle the effective ways of waste water.
Bacillus is a kind of aerobic or facultative anaerobic bacteria, can generate resistance endospore, is common Tiny ecosystem system
One of agent.The ectoenzyme that bacillus generates can decompose the organic matters such as starch, protein, fat, thus in water body of effectively degrading
Ammonia nitrogen, nitrite and sulfide etc..The Methylotrophic bacillus HB13000 that the present invention uses, is isolated from Hainan Province
Eight gulf mangrove bed muds have protein degradation matter, fat, starch and cellulolytic activity, livestock breeding wastewater can be effectively reduced
COD value, can be applied to the processing of livestock breeding wastewater.
Summary of the invention
The purpose of the present invention is to provide a kind of Methylotrophics with protein degradation matter, fat, starch and cellulose
Bacillus, the bacterial strain can be effectively reduced the COD value of livestock breeding wastewater, can be used as a kind of new useless for livestock and poultry cultivation
The microbial resources of water process.
To achieve the above object, the invention adopts the following technical scheme:
It is provided by the present invention with protein degradation matter, fat, starch and cellulolytic activity Methylotrophic brood cell's bar
Bacterium HB13000, on January 18th, 2016 in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC) culture presevation has been carried out, and has proved to survive, preservation registration number is CGMCC No.12064.Save address are as follows: Beijing
The institute of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica.The bacillus HB13000 belong to bacterium domain,
Firmicutes, bacillus guiding principle, bacillus mesh, Bacillaceae, Bacillus.
Methylotrophic bacillus HB13000 of the invention obtains process are as follows: acquires from Hainan Province Ba Menwan mangrove
Bed mud sample, sample use 80 DEG C of water bath processing 20min to kill non-brood cell bacterium after gradient dilution, are distributed in bacterium on the way thereafter
Isolation medium plate.The bacillus obtained again to separation carries out production protease, lipase, amylase and cellulase activity
Measurement, finishing screen select the bacillus HB13000 for being provided simultaneously with four kinds of enzymatic activitys.
The bacillus HB13000 has following biological characteristics: bacterial strain is crossed training on nutrient agar
After supporting 3d, bacterium colony is rounded, smooth wet, slightly swells；Thallus elongated rod shape, it is movable.Gram's staining, VP measurement, oxidizing ferment,
The catalase experiment positive, MR experiment is negative, using citrate, can restore nitrate, hydrolyzable starch, albumen, fat and fibre
Dimension element, can make gelatin liquefaction.
The 16S rDNA sequence of bacillus HB13000 is KU517813 in the preservation number of GenBank.It reflects through polyphase sort
It is set to Bacillus methylotrophicus (Methylotrophic bacillus).The bacterial strain is easy to grow, and has degradation egg
The COD value of waste water can be effectively reduced in livestock breeding wastewater processing, can develop for the ability of white, fatty, starch and cellulose
At a kind of new application in the microbial bacterial agent of cultivating wastewater purification.
Application of the bacillus HB13000 in livestock breeding wastewater processing, includes the following steps:
(1) bacterial strain activates: the Methylotrophic bacillus HB13000 is inoculated in nutrient agar, in
30 DEG C are cultivated for 24 hours, activated strains；Culture medium: 1~3g/L of beef extract, 5~10g/L of peptone, 1~5g/L of sodium chloride, agar powder
18~20g/L, pH6.5~7.5.
(2) Liquid Culture: the bacterial strain of activation is accessed in liquid seed culture medium, in 25~35 DEG C, 120~200r/min
Shaken cultivation 18~for 24 hours, seed liquor is made.Culture medium: 1~3g/L of beef extract, 5~10g/L of peptone, 1~5g/L of sodium chloride,
PH6.5~7.5.
(3) fermented and cultured: 5% being inoculated in liquid fermentation medium by volume for seed liquor, 25~35 DEG C, 120~
200r/min 48~72h of shaken cultivation, obtains the liquid bacterial agent of Methylotrophic bacillus HB13000.Culture medium: beef
1~3g/L of cream, 5~10g/L of peptone, 1~5g/L of sodium chloride, pH6.5~7.5.
(4) liquid bacterial agent is inoculated in 3%~10% inoculum concentration equipped with livestock breeding wastewater (adjusting pH 7.0 or so)
Conical flask in, 48~72h of shaking table culture under the conditions of 25~35 DEG C, 100~200r/min.
Experiment show to add bacillus HB13000CGMCC No.12064 can COD value effectively in degrading cultivation waste water,
When handling 48h, COD value is down to 295mg/L by the 3136mg/L originated, and COD removal rate reaches 90.6%；When handling 72h, COD
Value is down to 191mg/L, and COD removal rate reaches 93.9%.It can be seen that bacillus HB13000 CGMCC of the invention
No.12064 can be applied to the processing of livestock breeding wastewater.
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification.
Detailed description of the invention
Fig. 1 be bacterial strain HB13000 of the present invention morphological feature photo, × 1000.
Fig. 2 is the phylogenetic tree based on 16S rDNA of bacterial strain HB13000 of the present invention.
Specific embodiment
The present invention is further explained in the light of specific embodiments:
Embodiment 1: the separation of bacillus HB13000 CGMCC No.12064
Using 5 point samplings, bed mud sample is acquired from Hainan Province Ba Menwan mangrove.5g sample is taken to be dissolved in 45ml sterile
In 50% old seawater, it is placed in 28 DEG C, vibrates 30min in the constant-temperature table of 200r/min, is configured to 10-2~10-4Serial soil
Suspension.Use 80 DEG C of water bath processing 20min to kill non-brood cell bacterium after, draw serial suspension 0.1ml respectively, be coated on bacterium
Isolation medium is placed in 30 DEG C of culture carton upside down cultures.When bacterium colony is grown, according to colony shape, color, rim condition, thoroughly
Lightness, surface dry and wet state isophenous feature, the scribing line of picking single colonie purifying 2~3 times, until obtaining pure culture.Tentatively arranged
It after weight, numbers, saves.
The Species estimation of 2 bacterial strain HB13000 CGMCC No.12064 of embodiment
(1) form and cultural character
Bacterial strain is crossed on nutrient agar after culture 3d, bacterium colony it is smooth it is wet, slightly protuberance, round, edge is neat
It is whole.Thallus elongated rod shape, morphological features are shown in Fig. 1.
(2) physiological and biochemical property
Bacterial strain HB13000 can utilize glucose, maltose, sucrose, mannitol, fructose, lactose, xylose and citrate.
Gram's staining, VP measurement, oxidizing ferment, catalase experiment are positive, and MR experiment is negative, movable, can utilize citrate, can be also
Orthonitric acid salt can make gelatin liquefaction.
(3) 16S rDNA sequencing and Phylogenetic Analysis
The 16S rDNA sequence 1383bp of HB13000 is obtained by PCR amplification, sequencing, nucleotides sequence is listed in Genbank's
Accession number is KU517813.The sequence is compared with the sequence in EzBioCloud database, discovery bacterial strain HB13000 with
Bacillus methylotrophicus CBMB205T、Bacillus amyloliquefaciens subsp.plantarum
FZB42TWith highest 16S rDNA sequence similarity 99.93%.The related strain for selecting homology high is sharp in MEGA5.0
With Neighbor-joining method phylogenetic tree construction (Fig. 2), it can be seen that HB13000 and Bacillus
methylotrophicus CBMB205TIn same branch, the two affiliation is closer.Compare the two morphological feature, life
Biochemical character is managed, discovery morphological feature is similar, and physiological and biochemical property is consistent.
Based on the above results, bacterial strain HB13000 (CGMCC No.12064) of the present invention, is accredited as Bacillus
Methylotrophicus (Methylotrophic bacillus), on January 18th, 2016 in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center (CGMCC) has carried out culture presevation, and proves to survive, preservation registration number CGMCC
No.12064.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment 3: the preparation of bacterial strain HB13000 CGMCC No.12064 liquid bacterial agent
(1) bacterial strain activates: the Methylotrophic bacillus HB13000 is inoculated in nutrient agar solid culture
Base, for 24 hours in 30 DEG C of cultures, activated strains；Nutrient agar: beef extract 3g/L, peptone 10g/L, sodium chloride 5g/L, fine jade
18g/L, pH7.0,121 DEG C of cosmetics, 20min high pressure sterilization.
(2) Liquid Culture: the bacterial strain of step (1) activation is accessed in nutrient agar fluid nutrient medium, in 30 DEG C, 180r/
For 24 hours, seed liquor is made in min shaken cultivation.
(3) the seed fermentation liquid that step (2) is obtained 5% fermented and cultured: is inoculated in the training of nutrient agar liquid by volume
Support base, 30 DEG C, 180r/min shaken cultivation 48h.
The liquid bacterial agent of Methylotrophic bacillus HB13000 is obtained by above step.
Embodiment 4: bacterial strain HB13000 CGMCC No.12064 degradation of organic substances determination of activity
Using flat band method measurement bacterial strain HB13000 to protein, fat, starch, cellulose degrading activity, the results are shown in Table
1。
Produce protease assay: take single colonie, select be connected to protein hydrolyzing culture medium (culture medium form: skimmed milk power 50g,
Soluble starch 10g, yeast extract 5g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.2g, agar 20g, water 1000ml, pH 7.0), in 37
DEG C be inverted culture 3d, if bacterial strain generate protease, periphery of bacterial colonies have hydrolysis circle, hydrolytic circle/colony diameter ratio
(Hc value) has reacted the relative size of proteinase activity.The results show that bacterial strain HB13000 is living with stronger protein degradation matter
Property, Hc value is 2.80.
Yielding lipase measurement: taking single colonie, and point is connected to fat splitting culture medium (culture medium composition: lard 100g, peptone
10g, beef extract powder 8g, yeast extract 4g, glucose 20g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, sulfuric acid
Magnesium 0.2g, manganese sulfate 0.04g, agar 20g, water 1000ml, pH 6.5, micro dimethyl diaminophenazine chloride indicator), if 3d is cultivated in 37 DEG C of inversions
There is punctation appearance, then can determine that the bacterial strain has lipolytic ability.The results show that bacterial strain HB13000 has degradation rouge
The activity of fat.
It produces determination of amylase: taking single colonie, point is connected to Starch Hydrolysis culture medium, and (culture medium forms: peptone 10g, solvable
Property starch 2g, beef extract 5g, sodium chloride 5g, agar 20g, water 1000ml, pH7.0), after 37 DEG C of inversions culture 3d, into plate
Add Lugol's iodine solution.Iodine is met due to starch and becomes blue, if bacterial strain produces amylase, periphery of bacterial colonies can show colorless and transparent circle, transparent
Loop diameter/colony diameter ratio (Hc value) has reacted the relative size of amylase activity.The results show that bacterial strain HB13000 has
There is stronger degradable starch activity, Hc value is 3.25.
Cellulase-producing measurement: taking single colonie, and point is connected to sodium carboxymethylcellulose culture medium (culture medium composition: ammonium sulfate
2.0g, magnesium sulfate 0.5g, potassium dihydrogen phosphate 1.0g, sodium chloride 0.5g, sodium carboxymethylcellulose 20.0g, pH 7.2, agar
20.0g, water 1000ml), 3d are cultivated in 37 DEG C of inversions, with 1% congo red staining 30min, are decolourized 20min (2 with 1mol/L sodium chloride
It is secondary), if bacterial strain cellulase-producing, periphery of bacterial colonies has colorless and transparent circle, hydrolytic circle/colony diameter ratio (Hc value)
The relative size of cellulase activity is reacted.The results show that bacterial strain HB13000 has stronger degraded cellulose activity, Hc
Value is 4.30.
The inulinase-producing activity and enzyme activity of 1 bacterial strain HB13000 of table
Embodiment 5: bacterial strain HB13000 CGMCC No.12064 degrading waste water effect
Bacillus HB13000 CGMCC No.12064 is prepared as described in Example 3.Adjust wastewater pH to 7.0 or so,
It is added in the 250mL conical flask equipped with 100mL waste water by 5% inoculum concentration, the shaking table culture 72 under the conditions of 30 DEG C, 180r/min
Hour, during which timing sampling surveys COD, and calculates COD degradation rate.COD measurement uses potassium dichromate method, and the results are shown in Table 2.It is real
Test show to add bacillus HB13000 CGMCC No.12064 can COD value effectively in degrading cultivation waste water, after cultivating 48h
Reach requirement of the discharge standard to COD value.
2 bacterial strain of table handles the COD value variation of different time waste water
Sequence table
Sequence table (1383bp).
Claims (5)
1. a kind of bacillus, it is characterised in that the bacillus is Methylotrophic bacillus (Bacillus
Methylotrophicus), bacterial strain code name is HB13000, which is preserved in Chinese microorganism strain on January 18th, 2016
Preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Object research institute, culture presevation number are CGMCC NO.12064.
2. a kind of fermentation liquid of bacillus described in claim 1, which is characterized in that with the Methylotrophic bacillus
HB13000 is made through bacterial strain activation, seed culture, fermented and cultured, specifically includes the following steps:
(1) bacterial strain activates: the Methylotrophic bacillus HB13000 being inoculated in nutrient agar, in 30 DEG C
Cultivate 48~72h, culture medium: 1~3g/L of beef extract, 5~10g/L of peptone, 1~5g/L of sodium chloride, 18~20g/ of agar powder
L, pH6.5~7.5；
(2) it Liquid Culture: by the bacterial strain access liquid seed culture medium of activation, is vibrated in 25~35 DEG C, 120~200r/min
Culture 18~for 24 hours, seed liquor is made, culture medium: 1~3g/L of beef extract, 5~10g/L of peptone, 1~5g/L of sodium chloride,
PH6.5~7.5；
(3) seed liquor 5% fermented and cultured: is inoculated in liquid fermentation medium, 25~35 DEG C, 120~200r/ by volume
Min 48~72h of shaken cultivation, obtains the liquid bacterial agent of Methylotrophic bacillus HB13000, culture medium: beef extract 1~
3g/L, 5~10g/L of peptone, 1~5g/L of sodium chloride, pH6.5~7.5.
3. the application of bacillus described in claim 1, which is characterized in that the bacterium has protein degradation matter, fat, starch and fibre
Tie up the activity of element.
4. the application of bacillus described in claim 1, which is characterized in that the bacterium can be effectively reduced livestock breeding wastewater
COD value.
5. the application of bacillus described in claim 1, which is characterized in that the bacterial strain and its fermentation liquid are useless in livestock and poultry cultivation
Application in water process.
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| CN109207395B (en) * | 2018-09-11 | 2021-02-02 | 天津科技大学 | Methylophagus strain and application thereof |
| CN110452852B (en) * | 2019-08-26 | 2021-09-28 | 浙江省农业科学院 | Farmer market biomass waste fermentation inoculant and preparation method thereof |
| CN110923161A (en) * | 2019-11-12 | 2020-03-27 | 浙江理工大学 | Deodorizing probiotic preparation and preparation method and application thereof |
| CN115141770B (en) * | 2022-06-27 | 2023-08-29 | 安阳工学院 | Bacillus tequilensis H1 capable of degrading COD in livestock wastewater and application thereof |
| WO2024083600A1 (en) * | 2022-10-20 | 2024-04-25 | Basf Se | Odor controlling bacteria |
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