CN106635855B - Microbacterium and its culture application are seen in a kind of north - Google Patents
Microbacterium and its culture application are seen in a kind of north Download PDFInfo
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Abstract
The present invention relates to a kind of north to see microbacterium and its culture application.The bacterial strain be north see microbacterium (Microbacterium kitamiense) FSTB-4, it is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on June 1st, 2015, deposit number is CGMCC No.10939.The bacterial strain can be applied to the efficient removal of COD in the brine waste of salt content 1.0wt%-5.0wt%.The present invention also provides the salt tolerant microbial inoculum of de- COD a kind of, in the microbial inoculum include north see microbacterium (Microbacterium kitamiense) FSTB-4.The features such as COD in microbacterium removing high slat-containing wastewater capable of being fast degraded is seen in north provided by the invention, has salt resistance ability strong, high treating effect.
Description
Technical field
The invention belongs to field of environment microorganism, and in particular to a kind of de- north COD of salt tolerant sees that microbacterium and its culture are answered
With efficient removal of the bacterial strain especially suitable for COD in high slat-containing wastewater.
Background technique
From the industries such as petroleum, chemical industry, medicine and chemical fertilizer salt content higher than 1% high slat-containing wastewater generally contain compared with
High COD, Pollutants in Wastewater, which is constantly discharged into environment, can cause serious pollution problem.The shortage problem day of freshwater resources
Beneficial serious, the processing and its recycling of waste water are more valued by people.
The physically or chemically COD in method processing high slat-containing wastewater, it is at high cost and be likely to result in secondary pollution.Tradition is raw
Object method has great advantage when handling Low-salinity waste water, but in high salt, high soda acid, high temperature or low temperature inverse ring border, microorganism
Degradation capability will be suppressed.Facultative Halophiles, which handle high slat-containing wastewater, has the unrivaled advantage of conventional activated sludge process, and
It is more at low cost than physical-chemical process, more apparent economic benefit and social benefit can be generated, it can be up to standard for related wastewater treatment
Discharge provides technical support.
The problem of Facultative Halophiles technology is based on engineering field generates with being badly in need of, and it is high to tame out processing by scientific method
The advantage Facultative Halophiles of brine waste, the bacteroid can be in higher salinity with its unique eucaryotic cell structure and material composition
It is grown in environment.The salt-tolerance character of such salt-durable microbe with its adapt to extreme environment abundant enzyme system and activated product it is related.
In high slat-containing wastewater process field, salt content is a key factor for influencing microbiological treatment waste water efficiency.
Believe glad equal (biological reinforcing technology processing highly salt containing organic waste water [J] water treatment technology, 2008,8:66-70) using biological reinforced
Technical treatment highly salt containing organic waste water, using the dehydrogenation of the biological reinforced saponin waste water biological treatment system activated sludge of Facultative Halophiles
Enzymatic activity significantly improves, and when system tolerance chlorine ion concentration is up to 2.8%, the removal rate of saponin waste water COD is 84.41%.
CN201210130657.0, CN201210130644.3, CN201010536065.X and CN201210130658.5 are related to adopting
With marsh cock Salmonella (Kocuri apalustris) FSDN-A, Staphylococcus cohnis (Staphylococcus cohnii)
FSDN-C, arthrobacterium (Arthrobacter creatinolyticus) FDN-1, Shui Shi Flavobacterium (Flavobacterium mizutaii) FDN-2, Paracoccus denitrificans (Paracoccus denitrificans) DN-3 and Methylobacterium
(Methylobacterium phyllosphaerae) SDN-3 in brine waste and catalytic cracking catalyst waste water ammonia nitrogen,
The method that total nitrogen and COD carry out biological treatment.This method, which needs to cultivate a variety of bacterial strains and carries out compounding, prepares Facultative Halophiles
Agent, preparation process is complicated, and production cost is higher.
Currently, screening the relevant report that microbacterium is seen in the north for handling high slat-containing wastewater not yet.
Summary of the invention
The purpose of the present invention is to provide a kind of de- north COD of salt tolerant to see microbacterium and its culture application.The present invention provides
North see microbacterium can with fast degradation remove high slat-containing wastewater in COD, have salt resistance ability it is strong, the spies such as high treating effect
Point.
The present invention provides a kind of north and sees microbacterium FSTB-4, classification naming be north see microbacterium (Microbacterium kitamiense), it is preserved in " in China Committee for Culture Collection of Microorganisms's common micro-organisms on June 1st, 2015
The heart ", deposit number CGMCCNo.10939.
Microbacterium FSTB-4, main morphological features are seen in north provided by the invention are as follows: colony colour is shallower taupe brown, bacterium
Strain individual is rod-shaped.Physiological and biochemical property shows themselves in that Gram-positive, and oxidase negative contacts enzyme positive, can decompose using more
Kind carbon source, has nitrate reduction characteristic.The bacterial strain is resistant to aztreonam or acidum nalidixicum.
North provided by the invention sees that the 16SrRNA gene order of microbacterium FSTB-4 is shown in sequence table.
Application of the microbacterium FSTB-4 in brine waste removing COD is seen in north provided by the invention.The bacterial strain can be applied
The efficient removal of COD in the brine waste of salt content 1.0wt%-5.0wt%.
In the present invention, the brine waste be can be by pretreated chemical industry synthesis waste water, coal chemical industrial waste water, can also be with
It is that the waste water of the generations such as oil refining process, coal chemical industry technique and chemical industry synthesis technique passes through the dense salt of reverse osmosis minimizing unit generation
Water.The brine waste salt content is 0.5wt%-5.0wt%, preferably 1.0wt%-4.5wt%, COD(Cr method, similarly hereinafter) content
For 200-20000mg/L.
The cultural method of microbacterium FSTB-4, including bacterial strain activation, the culture of liquid seeds liquid, exposure are seen in north provided by the invention
Air culture, which is supported, waits three phases, specific steps are as follows:
(1) bacterial strain activates: north is seen that microbacterium FSTB-4 is inoculated into the inclined-plane or plate of FSTB solid medium,
25-35 DEG C is cultivated 48 hours, is stored in 4 DEG C of refrigerators.
(2) liquid seeds liquid culture: FSTB fluid nutrient medium is prepared, is sub-packed in triangular flask, is sterilized and be cooled to room temperature
Afterwards, the strain inoculated activated in picking inclined-plane or plate under gnotobasis is into triangular flask, 25-35 DEG C of culture 24-72h.It is described
FSTB fluid nutrient medium are as follows: FeSO4•7H2O 25mg/L, NH4NO3286mg/L, KCl 929mg/L, CaCl22769mg/L,
NaCl 21008mg/L, beef extract 5g/L, peptone 10g/L, pH value range 6.0-8.5, preferably 6.5-8.0.FSTB is solid
Body culture medium is that 20g/L agar is added in liquid medium.
(3) aeration culture: being added FSTB fluid nutrient medium in the reactor for being equipped with aerator or saliferous to be processed is useless
Water is inoculated with liquid seed liquor according to the ratio of volume ratio 5%-25%, and pH value control is cultivated 48-96 hours in 6.0-8.5, aeration,
Periodic feed supplement and discharge operation are carried out later, and withdrawal rate accounts for the 5%-90% of reactor volume, and feed supplement amount accounts for reactor volume
5%-90%, can also add a small amount of carbon source, nitrogen source and microelement substance, cultivate 24-48 hour as 1 cultivation cycle, it
Afterwards according to the culture solution of aforementioned proportion discharge corresponding volume, thus obtain seeing containing higher concentration north that the dense bacterium solution of microbacterium produces
Product.
In the present invention, after culture, the bacterium solution that acquisition culture finishes is applied to containing FSTB solid medium after dilution
Plate in, the percentage of bacterium colony similar to FSTB-4 is counted and counted to the bacterium colony grown.In closed culture environment
Under, north sees that microbacterium is pure bacterial strain, and percent similarity reaches 95% or more.It cultivates the growing environment of FSTB-4 and continues although having
The selection pressure with high salt of property, but still might have a small amount of other interference strain growths in long term open formula culture, to prevent
It interferes bacterium duration flourish to become dominant bacteria, when percent similarity is close to 50%, intakes again after whole discharges can be taken
The mode of inoculated and cultured restores normal open culture operating process later.
It include that microbacterium FSTB-4 is seen in north the present invention also provides the salt tolerant microbial inoculum of de- COD a kind of, in the microbial inoculum.Described is resistance to
Salt microbial inoculum, which can be, sees salt tolerant microbial inoculum made from microbacterium FSTB-4 bacterium solution only with north, can also be multiple with other salt tolerant microbial inoculums
Match, can such as be realized preferably useless in conjunction with the existing microbial bacterial agent with denitrogenation, dephosphorization and hardly degraded organic substance of degrading
Water treatment efficiency.
Salt tolerant north provided by the invention sees that microbacterium FSTB-4 is one plant and has preferable COD detachability in Halite water body
The bacterial strain of energy, salt resistance ability is strong, high treating effect, is used directly for the processing of brine waste, can also be added to existing contain
In salt waste water treatment system, water treatment effect is improved.The cultural method of microbacterium FSTB-4 is seen in north provided by the invention, can be straight
It connects and is carried out using sewage treatment plant's brine waste (adding a small amount of natural medium and chemical synthesis culture medium according to condition of water quality)
Dominant strain culture is enriched with enrichment work, greatly maintains the growth characteristics and resistance characteristics of salt tolerant microbial inoculum, and reduces Facultative Halophiles
The toxigenic capacity of agent.
Biomaterial preservation explanation
North provided by the invention see microbacterium (Microbacterium kitamiense) FSTB-4 bacterial strain, it is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC)；Address: BeiChen West Road, Chaoyang District, BeiJing City 1
No. 3 Institute of Microorganism, Academia Sinica, institute；Deposit number: CGMCC No.10939；Preservation date: on June 1st, 2015.
Specific embodiment
North of the invention sees that microbacterium FSTB-4 is in June, 2014 from being derived from Hunan Yueyang petroleum chemical enterprise for from
Separation screening obtains in the activated sludge of reason epoxychloropropane production waste water, and physiological and biochemical test the results are shown in Table 1.
The physiological and biochemical test result of microbacterium FSTB-4 is seen in 1 north of table
It is identified through " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", which belongs to north and see microbot
Bacterium (Microbacterium kitamiense), it is named as FSTB-4.
Embodiment 2 is screened and is isolated and purified
It is used to handle ring to Hunan Yueyang petroleum chemical enterprise is derived from using the method that culture solution salinity is gradually increased
The activated sludge of oxygen chloropropane production waste water carries out domestication culture 15 days.
The activated sludge for taking above-mentioned domestication good carries out initial gross separation using dilution spread method in enriched medium, by
This obtains 15 bacterium colonies that growth conditions are good and character is different.The main component that used enriched medium contains are as follows: COD
For 1000mg/L, calcium ion concentration 1000mg/L, total salt quantity 10000mg/L, agar contains in used solid medium
Amount is 2.0%.
Then it is transferred using plate streaking method to obtained bacterium colony, scribing line is transferred in screening and culturing medium, is carried out
Further separation and screening.The main component that the screening and culturing medium of use contains are as follows: COD 1000mg/L, calcium ion concentration are
4000mg/L, total salt quantity 25000mg/L, agar content is 2.0% in used solid medium.
10 plants of pure bacterial strains are obtained by aforesaid operations, wherein one plant is that microbacterium FSTB-4 is seen in north.
The investigation of microbacterium FSTB-4 salt resistance ability is seen in 3 north of embodiment
North of the present invention sees that the cultural method of microbacterium FSTB-4 includes: bacterial strain activation, the culture of liquid seeds liquid, exposes
Air culture is supported, and detailed process is as follows:
(1) bacterial strain activates: north being seen that microbacterium FSTB-4 is inoculated into the plate containing FSTB solid medium, 30 DEG C of rings
It cultivates 48 hours, is then stored in 4 DEG C of refrigerators stand-by under border.The FSTB solid medium are as follows: FeSO4•7H2O 25mg/L,
NH4NO3286mg/L, KCl 929mg/L, CaCl22769mg/L, NaCl 21008mg/L, beef extract 5g/L, peptone 10g/
L, agar 20g/L, pH value 7.8.
(2) liquid seeds liquid culture: FSTB fluid nutrient medium is prepared, is sub-packed in triangular flask, is sterilized and be cooled to room temperature
Afterwards, it is small that 48 are cultivated under gnotobasis into triangular flask, under the conditions of 30 DEG C with the strain inoculated after activation in oese picking plate
When.The FSTB fluid nutrient medium are as follows: FeSO4•7H2O 25mg/L, NH4NO3286mg/L, KCl 929mg/L, CaCl2
2769mg/L, NaCl 21008mg/L, beef extract 5g/L, peptone 10g/L, pH value 7.8.
(3) it aeration culture: is cultivated using closed reactor, used reactor and various utensils are required to sterilize
Completely, air inlet and exhaust position need to install bacteriological filter apparatus, and culture solution, acid-base modifier and trace element solution are required to go out
It is added after bacterium according to sterile working regulation, reactor need to be equipped with aerator, and can be intake, adjust acid, alkali tune, feed supplement and row
The operation of water discharge, the FSTB fluid nutrient medium after being put into sterilizing in the reactor, according to the ratio of reactor volume percentage 10%
Example inoculation liquid seed liquor, after opening incubation, uses soda acid automatic control system by medium pH value model in incubation
System is contained in 6.0-8.5, aeration culture carries out periodic feed supplement and discharge operation after 72 hours, discharge is reactor volume
The culture solution of percentage 50%, feed supplement are the FSTB fluid nutrient medium of reactor volume percentage 50%, can also be added simultaneously a small amount of
Carbon source, nitrogen source and microelement substance, culture 24 hours be 1 cultivation cycle, later according to aforementioned proportion be discharged corresponding body
Thus long-pending culture solution obtains the dense bacterium solution product that microbacterium is seen containing pure north.
The dense bacterium solution that above-mentioned culture finishes is acquired, is applied to after dilution in the plate containing FSTB solid medium, to length
Bacterium colony out is counted and counts the percentage of bacterium colony similar to FSTB-4, and percent similarity reaches 95% or more, that is, assert should
North sees that microbacterium is pure bacterial strain.
See that microbacterium FSTB-4 handles brine waste using the north that above-mentioned culture obtains.The present embodiment is by pretreated
For epichlorohydrin production process brine waste, the water quality of handled brine waste are as follows: COD concentration range is 800-
2000mg/L, salt content 1wt%-5wt%, treatment temperature are 30 DEG C.Treatment effect is as shown in table 2.
The effect of microbacterium FSTB-4 processing brine waste is seen in 2 north of table
As shown in Table 2, north sees that the salt content that microbacterium FSTB-4 is resistant to is 1.0wt%-5.0wt%, can efficiently take off
Except the COD in waste water.
The open culture of microbacterium FSTB-4 is seen in 4 north of embodiment
North of the present invention sees that the open culturing method of microbacterium FSTB-4 includes: bacterial strain activation, the culture of liquid seeds liquid, opening
The culture of formula aeration, the detection of pure bacterial strain and dominant bacteria maintenance, detailed process is as follows:
(1) bacterial strain activates: north being seen that microbacterium FSTB-4 is inoculated into the plate containing FSTB solid medium, 30 DEG C of rings
It cultivates 48 hours, is then stored in 4 DEG C of refrigerators stand-by under border.The FSTB solid medium are as follows: FeSO4•7H2O 25mg/L,
NH4NO3286mg/L, KCl 929mg/L, CaCl22769mg/L, NaCl 21008mg/L, beef extract 5g/L, peptone 10g/
L, agar 20g/L, pH value 7.8.
(2) liquid seeds liquid culture: FSTB fluid nutrient medium is prepared, is sub-packed in triangular flask, is sterilized and be cooled to room temperature
Afterwards, it is small that 48 are cultivated under gnotobasis into triangular flask, under the conditions of 30 DEG C with the strain inoculated after activation in oese picking plate
When.The FSTB fluid nutrient medium are as follows: FeSO4•7H2O 25mg/L, NH4NO3286mg/L, KCl 929mg/L, CaCl2
2769mg/L, NaCl 21008mg/L, beef extract 5g/L, peptone 10g/L, pH value 7.8.
(3) brine waste to be processed, this reality open culture: is added in the open reactive device for being equipped with aerator
Example is applied for by pretreated epichlorohydrin production process brine waste, the water quality of handled brine waste are as follows: COD is dense
Degree range is 1000mg/L, and salt content 1wt%-5.0wt%, cultivation temperature is 35 DEG C, according to the ratio inoculation liquid of volume ratio 10%
Body seed liquor, aeration culture 96 hours, culture solution cell concentration changes (absorption values OD when with 600nm600Indicate thallus
Concentration) as shown in table 3.
3 north of table sees that microbacterium FSTB-4 cell concentration changes
(4) pure bacterial strain detection: the bacteria agent mixed liquor that above-mentioned culture finishes is acquired, is applied to after dilution solid containing FSTB
In the plate of body culture medium, the percentage of bacterium colony similar to FSTB-4, similar percentage are counted and counted to the bacterium colony grown
Than reaching 50% or more, then assert that north sees that microbacterium is dominant strain.
(5) dominant strain is safeguarded: when the pure bacterial strain similar bacterium colony percentage of detection FSTB-4 is close to 50%, taking whole rows
Again water inlet and the again mode of inoculated and cultured after material restore normal open culture process later.
North of the present invention sees that microbacterium FSTB-4 is very good to the adaptability and treatment effect of practical brine waste, has
Stronger COD degradation function can be individually used for the processing of brine waste using the pure culture of the bacterial strain, be made after can also amplifying culture
It is added to prior art process for external source functional microorganism, adjusting targetedly is optimized to actual process situation, is solved
Active sludge treatment effect difference and easy the problem of degenerating under hypersaline environment.Physical chemistry is compared using Facultative Halophiles processing high slat-containing wastewater
Method is at low cost, and directly application can generate more apparent economic benefit and social benefit, can reach for high saliferous correlation wastewater treatment
Mark discharge provides technical support.
SEQUENCE LISTING
<110>Sinopec Group
Dalian Petroleum Chemical Engineering Institute, Sinopec Group
<120>microbacterium and its culture application are seen in a kind of north
<130>new application
<170> PatentIn version 3.5
<211> 1304
<212> DNA
<213> Microbacterium
aacgggtgag taacacgtga gcaacctgcc ctgaactctg ggataagcgc tggaaacggc 60
gtctaatact ggatatgagc tgcggaggca tcttcgtggt tggaaagatt tttcggtttg 120
ggatgggctc gcggcctatc agcttgttgg tgaggtaatg gctcaccaag gcgtcgacgg 180
gtagccggcc tgagagggtg accggccaca ctgggactga gacacggccc agactcctac 240
gggaggcagc agtggggaat attgcacaat gggcggaagc ctgatgcagc aacgccgcgt 300
gagggatgac ggccttcggg ttgtaaacct cttttagcag ggaagaagcg agagtgacgg 360
tacctgcaga aaaagcgccg gctaactacg tgccagcagc cgcggtaata cgtagggcgc 420
aagcgttatc cggaattatt gggcgtaaag agctcgtagg cggtttgtcg cgtctgctgt 480
gaaatctggg ggctcaaccc ccagcctgca gtgggtacgg gcagactaga gtgcggtagg 540
ggagattgga attcctggtg tagcggtgga atgcgcagat atcaggagga acaccgatgg 600
cgaaggcaga tctctgggcc gtaactgacg ctgaggagcg aaagggtggg gagcaaacag 660
gcttagatac cctggtagtc caccccgtaa acgttgggaa ctagttgtgg ggtccattcc 720
acggattccg tgacgcagct aacgcattaa gttccccgcc tggggagtac ggccgcaagg 780
ctaaaactca aaggaattga cggggacccg cacaagcggc ggagcatgcg gattaattcg 840
atgcaacgcg aagaacctta ccaaggcttg acatacacga gaacgctgca gaaatgtaga 900
actctttgga cactcgtgaa caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 960
ttgggttaag tcccgcaacg agcgcaaccc tcgttctatg ttgccagcac gtaatggtgg
1020
gactcatggg atactgccgg ggtcactcgg aggaaggtgg ggatgacgtc aaatcatcat
1080
gccccttatg tcttgggctt cacgcatgct acaatggccg gtacaaaggg ctgcgatacc
1140
gcgaggtgga gcgaatccca aaaagccggt cccagttcgg attgaggtct gcaactcgac
1200
ctcatgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga atacgttccc
1260
gggtcttgta cacaccgccc gtcaagtcat gaaagtcggt aaca 1304
Claims (8)
1. microbacterium FSTB-4 is seen in a kind of north, classification naming be north see microbacterium (Microbacterium kitamiense),
It is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on June 1st, 2015, deposit number is
CGMCC No.10939。
2. application of the microbacterium FSTB-4 in brine waste removing COD is seen in north described in claim 1.
3. application according to claim 2, it is characterised in that: the bacterial strain can be applied to salt content 1.0wt%-5.0wt%
Brine waste in COD efficient removal.
4. application according to claim 2 or 3, it is characterised in that: the brine waste is closed by pretreated chemical industry
The waste water generated at waste water, coal chemical industrial waste water or oil refining process, coal chemical industry technique and chemical industry synthesis technique is by reverse osmosis
The strong brine that minimizing unit generates, salt content 0.5wt%-5.0wt%, COD content are 200-20000mg/L.
5. the cultural method of microbacterium FSTB-4 is seen in north according to claim 1, it is characterised in that including bacterial strain activation, liquid
Seed liquor culture, aeration culture three phases, specific steps are as follows:
(1) bacterial strain activates: north being seen that microbacterium FSTB-4 is inoculated into the inclined-plane or plate of FSTB solid medium, 25-35
DEG C culture 48 hours, be stored in 4 DEG C of refrigerators；
(2) liquid seeds liquid culture: preparing FSTB fluid nutrient medium, be sub-packed in triangular flask, after sterilizing and being cooled to room temperature, nothing
The strain inoculated activated in picking inclined-plane or plate under collarium border is into triangular flask, 25-35 DEG C of culture 24-72h；
(3) aeration culture: being added FSTB fluid nutrient medium or brine waste to be processed in the reactor for being equipped with aerator,
It is inoculated with liquid seed liquor according to the ratio of volume ratio 5%-25%, pH value control is aerated culture 48-96 hours, later in 6.0-8.5
Periodic feed supplement and discharge operation are carried out, withdrawal rate accounts for the 5%-90% of reactor volume, and feed supplement amount accounts for the 5%- of reactor volume
90%, culture 24-48 hours are 1 cultivation cycle, later according to the culture solution of aforementioned proportion discharge corresponding volume, are contained
The dense bacterium solution product of microbacterium is seen in higher concentration north.
6. according to the method described in claim 5, it is characterized by: the FSTB fluid nutrient medium are as follows: FeSO4•7H2O 25mg/
L, NH4NO3286mg/L, KCl 929mg/L, CaCl22769mg/L, NaCl 21008mg/L, beef extract 5g/L, peptone
10g/L, pH value 6.0-8.5；FSTB solid medium is that 20g/L agar is added in liquid medium.
7. according to the method described in claim 5, it is characterized by: in long term open formula culture, to prevent interference bacterium from continuing
Sexual reproduction is grown to serve as dominant bacteria, and acquisition culture finishes bacterium solution, is applied in the plate containing FSTB solid medium after dilution,
The percentage that bacterium colony similar to FSTB-4 is counted and counted to the bacterium colony grown is taken when percent similarity is close to 50%
The mode for inoculated and cultured of intaking again after whole discharges restores normal open culture operating process later.
8. a kind of salt tolerant microbial inoculum of de- COD, it is characterised in that: in the microbial inoculum include north see microbacterium (Microbacterium kitamiense) FSTB-4.
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| CN110229766B (en) * | 2019-06-14 | 2021-06-08 | 浙江工业大学 | Microbacterium oxydans and application thereof in degradation of organic pollutants |
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