CN106047768B - A kind of pottery luer bacteria strain and its application - Google Patents
A kind of pottery luer bacteria strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of pottery luer bacteria strain and its applications, belong to environmental microorganism application field, the Strain Designation is pottery strategic point Salmonella (Thauera sp.) CM-HZX2, now it is deposited in China typical culture collection center (abbreviation CCTCC), deposit number is CCTCC NO:M 2015689, and the deposit date is in November, 2015.Pottery strategic point Salmonella CM-HZX2 provided by the invention is a kind of efficient flocculant producing strains, be can be widely applied in sanitary sewage, especially the water body of high-turbidity water and raw water pollution；It is in pH 7.0, and 30 DEG C of temperature, revolving speed is grown most preferably under conditions of being 150r/min, and after liquid shake culture 72h, flocculating rate reaches as high as 92.3%.
Description
Technical field
The present invention relates to environmental microorganism application field more particularly to a kind of pottery luer bacteria strain and its applications.
Background technique
Flocculant is broadly divided into inorganic flocculating agent, organic polymer coargulator and microbial flocculant.Wherein inorganic flocculation
Agent China it is current account for important share in the market, have ten tens of thousands of tons of demand every year, mainly using molysite, aluminium salt as representative.
Wherein, molysite has corrosivity, residual easy to form, and the water that makes that treated causes secondary pollution with color；It defends
Raw learn finds that aluminium salt has direct relationship with current senile dementia in the intracorporal accumulation of people.Macromolecule organic flocculating agent, such as
Polyacrylamide etc., itself does not have toxicity, but is not easily decomposed, and long-term accumulation will also result in secondary pollution in nature, and third
Acrylamide monomer has very strong " three cause " effect, and yield of the polyacrylamide in 2006 in China has been up to more than 30 ten thousand tons,
It is environmentally safe using meeting on a large scale and human health cause to seriously affect.
Microbial flocculant (MBF) is that the one kind for being metabolized generation during the growth process by flocculating microbial has flocculation activity
High-molecular compound, main component is protein, polysaccharide, cellulose, DNA and thallus with flocculant activity etc.；It is
Using biotechnology, novel, efficient, the cheap water treatment agent of one kind as obtained from microculture, separation and Extraction.Micro- life
(microbe species that can generate agglutinating matter are various, including bacterium, actinomyces, saccharomycete and mould for object flocculant；It can gather
Suspended particulate, metal ion, pigment and organic substance in collection, precipitating water, with good, the biodegradable spy of flocculating effect
Property, it is nontoxic to environment and the mankind, secondary pollution will not be generated, be a kind of flocculant of highly effective and safe, thus increasingly by
To attention (the progress Agriculture of Anhui section of Yang Zhengliang, Zheng Xuebin, Feng Guiying microbial flocculant of domestic and international researcher
It learns, 2007,35 (24): 7593-7594,7596).
Currently, existing research person develops a series of microbial flocculants, some have been applied to related wastewater treatment and
In industrial treatment.The country is also constantly carrying out the research of microbial flocculant in recent years, and relevant research report is also very much,
But without reaching large-scale industrial production and application stage, it is still necessary to constantly carry out in a deep going way.Meanwhile microbial flocculant
High production cost, narrow single bacterium for producing flocculant application surface is the bottleneck problem for limiting its industrialized production and application.
The flocculation of microbial flocculant is mainly by solable matter, suspended particulate and the colloidal substance etc. in water
The modes such as biological adsorption bridge formation, electrical neutralization, crossing net collection are carried out to realize.The strain characteristic of bacterium for producing flocculant determines its institute
Produce the physics and chemistry of flocculant.
In order to reduce the production cost of microbial flocculant, microbial flocculant efficiently produces the separation screening of bacterium, utilizes
High COD/ high N waste water becomes studied both at home and abroad at present as the research directions such as Optimum Regulation of substitutive medium, fermentating metabolism
Hot spot.The strain of reported producing microbial flocculant has tens kinds, as Corynebacterium glutamicum,
Enterobacter aerogenes, Bacillus sp.F19, Bacillus sp.DP-152 etc., but only Kurane etc.,
In the bacterial strain that the rhodococcus erythropolis (Rhodococcus erythropolis) of discovery in 1986 is unique industrial applications, strain
Scarcity of resources, therefore, the screening of microbial flocculant superior strain are always one of the hot spot work in the field.
The critical issue for restricting microbial flocculant development at present is that production cost is excessively high and low output, therefore screens high
Imitating bacterium for producing flocculant and improving its flocculation efficiency becomes particularly important, while being also exploitation and system research microbial flocculant
The crucial first step.Therefore the bacterium with efficient producing microbial flocculant is filtered out, for processing high-turbidity water and original
The water body of water pollution has great utilization and extention value.
Summary of the invention
The present invention provides a kind of pottery luer bacteria strain and its application, which is that efficient flocculant generates
Bacterium can be widely applied in sanitary sewage, especially the water body of high-turbidity water and raw water pollution.
A kind of pottery luer bacteria strain is named as pottery strategic point Salmonella (Thauera sp.) CM-HZX2, deposit number CCTCC
NO:M 2015689, the deposit date is in November, 2015.
The pottery luer bacteria strain is in the Chinese Typical Representative culture of Wuhan University, preservation in November, 2015 Wuhan, China city
Collection (abbreviation CCTCC), deposit number are CCTCC NO:M 2015689, are named as pottery Salmonella (Thauera sp.) in distress
CM-HZX2。
The 16S rDNA sequence of described pottery strategic point Salmonella (Thauera sp.) CM-HZX2 is as shown in SEQ ID NO.1.
The biological property of described pottery strategic point Salmonella (Thauera sp.) CM-HZX2 are as follows: bacterium colony surface is round, wet, light
Yellow, it is transparent, be easy to provoke, neat in edge, Gram's staining reaction is negative.
It is described pottery strategic point Salmonella (Thauera sp.) CM-HZX2 physiological and biochemical property: oxidizing ferment, urease, arginine,
Lysine, ornithine, aesculin are positive, hydrogen sulfide production test, nitrate reduction, glucose fermentation, indoles, V.P, MR-VP, bright
It is feminine gender that glue, glycerol, which produce acid, sorbierite, mannitol, citric acid, salicin, phenylalanine experimental result,.
The present invention provides a kind of cultural methods of luer bacteria strain of making pottery to connect after activating the pottery luer bacteria strain
Kind is into the culture medium that initial pH value is 6~9, temperature is 25~35 DEG C, the mass concentration of salt is 0~5%, 150~200r/
It is cultivated under min revolving speed.The pottery luer bacteria strain growth activity for cultivating acquisition under this condition is high.
The present invention provides a kind of application of pottery luer bacteria strain in processing sanitary wastewater or industrial wastewater.
It is further preferred that the sanitary wastewater is high-turbidity water or the water body that raw water pollutes.
Application of the pottery luer bacteria strain in processing sanitary sewage waste water, comprising: by the pottery luer bacteria strain
After immobilization, then biochemical treatment is carried out in waste water.
The pottery luer bacteria strain immobilization includes: bacterial strain to be immobilized onto microballoon, or carry out bio-film colonization for bacterium
Strain is fixed on filler；When waste water passes through the microballoon or filler that are fixed with bacterial strain, bacterial strain is to solid difficult to degrade small in waste water
Grain is handled.
The present invention provides a kind of microbial inoculums comprising the pottery luer bacteria strain.
The present invention also provides a kind of microbe microspheres comprising the pottery luer bacteria strain.
The main component of carrier is the mixture of polyvinyl alcohol and sodium alginate in the microbe microsphere；Mass-transfer performance
It is good, and the stability of microbe microsphere is strong.
In order to enhance the stability of microbe microsphere, it is usually additionally added suitable active carbon, boric acid, calcium chloride in the carrier
Deng.
Active carbon can increase microballoon specific gravity, weaken Float upward function；Boric acid can increase microballoon specific surface and Kong Rong, internal
Pore structure be conducive to reaction substrate and nutriment transmitting and microbial cell carrier inside apposition growth；Chlorine
The permeability of microbe microsphere can be enhanced by changing calcium, improve activity.
The present invention also provides application of the microbial inoculum in processing sanitary wastewater or industrial wastewater.
The present invention also provides application of the microbe microsphere in processing sanitary wastewater or industrial wastewater.
Pottery strategic point Salmonella CM-HZX2 provided by the invention is a kind of efficient flocculant producing strains, and it is dirty to can be widely applied to life
In water, especially high-turbidity water and raw water pollution water body；It is in pH 7.0, and 30 DEG C of temperature, revolving speed is the item of 150r/min
It is grown most preferably under part, after liquid shake culture 72h, flocculating rate reaches as high as 92.3%.
Detailed description of the invention
Fig. 1 is the microscope photograph of present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2.
Fig. 2 is that present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2 is based on what 16S rDNA sequence homology constructed
System development tree.
Fig. 3 is influence of the initial pH value to present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2 growth.
Fig. 4 is influence of the temperature to present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2 growth.
Fig. 5 is influence of the revolving speed to present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2 growth.
Fig. 6 is influence of the salinity to present invention pottery strategic point Salmonella (Thauera sp.) CM-HZX2 growth.
Specific embodiment
The characterization of flocculating effect is with the following method: preparing the aqueous suspension ofkaolin of 4g/L, 50mL is taken to be placed in the ratio of 50mL
10% (mass fraction) CaCl is added in colour tube2Solution 2mL and fermentation liquid 1mL is adjusted with 1mol/L NaOH and 1mol/L HCl
PH value covers grinding port plug, colorimetric cylinder is made about 10 times and is overturn naturally, bubble rises when revolving speed to overturn every time to the value of setting
Subject to finishing；After overturning, 10min is stood.It takes supernatant to measure OD 550nm using spectrophotometry, is denoted as A.To be not added
Fermentation liquid adds the identical experiment of the other conditions of 1ml distilled water as control, measures the OD550nm (D 550) of corresponding supernatant,
It is denoted as B, then flocculating rate is pressed examination and calculated: η=(A-B) × 100/A.
Embodiment 1 is made pottery the separation and identification of Salmonella in distress (Thauera sp.) CM-HZX2
One, the separation of pottery strategic point Salmonella (Thauera sp.) CM-HZX2
Strategic point Salmonella (Thauera sp.) CM-HZX2 that makes pottery is separated from the activated sludge of Huzhou sewage treatment plant aerobic tank
It obtains.
(1) culture medium
LB culture medium: tryptone: 10gL-1, yeast powder: 5gL-1, NaCl:10gL-1, matched with no phenol distilled water
System, adjusting pH value are 7.0,121 DEG C of high-temp steam sterilizing 20min.10~20gL of addition is needed in LB Agar Plating-1's
Agar.
Fermentation medium: glucose: 6.0gL-1, yeast powder: 4.0gL-1, K2HPO4: 5.0gL-1, KH2PO4:
2.0g·L-1, NaCl:0.1gL-1, MgSO4: 0.2gL-1, prepared with distilled water, adjusting pH value is that 7.0,115 DEG C of high temperature steam
Vapour sterilizing 20min.
(2) bacterial strain isolates and purifies
It takes and is fetched in right amount from Huzhou sewage treatment plant, Shaoxing Wastewater Treatment Plant, Yong Quan printing and dyeing mill, printing and dyeing mill, U.S. Xingda and other places
Activated sludge sample (about 5g) be added in LB culture medium in batches, the shaken cultivation on 30 DEG C, the shaking table of 150r/min, after 3d
The bacterium solution 1mL for taking above-mentioned culture, is configured to 10-1、10-2、10-3、10-4、10-5、10-6Dilution, take 100~200 μ L plates
Be coated in solid medium tablets, each dilution does three repetitions, 30 DEG C constant temperature dark culturing 1 week, observe bacterium on plate
The growing state fallen.Wherein high-visible single colonie separating for several times purifying is selected, 123 plants of pure bacterial strains are obtained.
Primary dcreening operation: by strain inoculated after activation to being equipped in the triangular flask of 50mL screening and culturing medium 250mL, 30 DEG C, 120r/
Gained culture solution is carried out flocculation activity Preliminary Determination by mim shaken cultivation 72h.
Measuring method: in the 100mL liquid of 4g/L aqueous suspension ofkaolin, adding 2mL culture solution, and range estimation sees whether to make
Aqueous suspension ofkaolin flocculates into biggish floccule body, and with not plus culture solution is compared referring to sample, with this obtains primary dcreening operation bacterial strain.
Secondary screening: there will be the strain inoculated of flocculation activity to three of the 250mL equipped with 50mL screening and culturing medium obtained by primary dcreening operation
In the bottle of angle, 28 DEG C, 120r/min shaken cultivation 72h, gained culture solution is carried out to the measurement of flocculation activity, so that secondary screening is carried out,
Using flocculating rate size as measurement index.
8 plants of bacterial strains that secondary screening is obtained are centrifuged under 5000r/min after taking a certain amount of bacterium solution to be transferred to LB culture medium 72h
10min, supernatant is spare, measures the flocculating rate of fermenation raw liquid, supernatant in waste water, obtains one plant of best bacterial strain of effect,
It is named as CM-HZX2.
Two, the identification of pottery strategic point Salmonella (Thauera sp.) CM-HZX2
(1) bacterium colony of pottery strategic point Salmonella (Thauera sp.) CM-HZX2, thalli morphology observation
Thalli morphology research: Gram's staining is carried out to thallus, is seen under 16 × 100 eyepiece of optical microscopy
It examines.
The bacterial strain main biological property are as follows: it is aerobic, 35 DEG C under conditions of, by bacterial strain CM-HZX2 on LB culture medium
Culture for 24 hours after, bacterium colony surface be it is round, wet, faint yellow, transparent, be easy to provoke, neat in edge.Gram's staining reacts
It is negative.
The physiological and biochemical property of the bacterial strain: oxidizing ferment, urease, arginine, lysine, ornithine, aesculin are positive,
Hydrogen sulfide production test, nitrate reduction, glucose fermentation, indoles, V.P, MR-VP, gelatin, glycerol produce acid, sorbierite, mannitol,
Citric acid, salicin, phenylalanine experimental result are feminine gender.
(2) molecular biology identification of pottery strategic point Salmonella (Thauera sp.) CM-HZX2
The DNA that CM-HZX2 bacterial strain is extracted with the enzyme that Invitrogen (Shanghai) Trading Co., Ltd. synthesizes, expands the bacterial strain
The PCR product of acquisition is entrusted Shanghai Li Fei Bioisystech Co., Ltd to be sequenced by 16S rDNA after purification.
Primer is 1492R:5 '-GGY TAC CTT GTT ACG ACTT-3 '；27F:5 '-AGA GTT TGA TCM TGG
CTCAG-3 ' (Invitrogen's synthesis).
PCR amplification system is as shown in table 1.
1 PCR amplification system of table
PCR reaction condition are as follows:
The 16SrDNA segment that length is 1381bp is acquired through being sequenced, the number of registration in GenBank is
KT894041, base sequence is as shown in SEQ ID NO.1.By BLAST compare discovery CM-HZX2 16S rDNA with
The similitude of 23 plants of Thauera bacteriums such as Thauera sp.R-24450, Thauera sp.G3DM-88 is 99%.With
MEGA5.0 software constructs phylogenetic tree to these bacteriums, and carries out homology analysis.
As shown in Fig. 2, CM-HZX2 bacterial strain and the genetic distance of Thauera bacterium are nearest, in conjunction with the morphological feature of bacterial strain,
It can primarily determine that CM-HZX2 bacterial strain for strategic point Salmonella (Thauera sp.) of making pottery, is named as Thauera sp.CM-HZX2, and should
Bacterial strain send to the China typical culture collection center (abbreviation CCTCC) for being located at Wuhan, China university and carries out preservation, deposit number
For CCTCC NO:M 2015689, the deposit date is in November, 2015.
Embodiment 2 make pottery Salmonella in distress (Thauera sp.) CM-HZX2 the best growing condition optimization
Culture medium: LB culture medium tryptone: 10gL-1, yeast powder: 5gL-1, NaCl:10gL-1, steamed with no phenol
Distilled water is prepared, and adjusting pH value is 7.0,121 DEG C of high-temp steam sterilizing 20min.
(1) initial pH value of medium influences Salmonella (Thauera sp.) CM-HZX2 growth activity in distress of making pottery
By changing pH in culture medium value, initial p H is investigated to Salmonella (Thauera sp.) CM-HZX2 growth activity in distress of making pottery
Influence.The initial pH for adjusting culture medium is respectively 4.0,5.0,6.0,7.0,8.0,9.0,10.0, at 30 DEG C, 150r/min item
Shaken cultivation 72h under part measures bacterial growth amount, does blank with pottery strategic point Salmonella (Thauera sp.) CM-HZX2 is not added,
Separated in time sample detection OD600nm value.
As can be seen from Figure 3, the bacterium initial pH be 5.0 culture mediums below in, light absorption value is very low, slow growth, when pH by
5.0 when increasing to 6.0, and OD600nm light absorption value rapidly increases, and in 6.0-9.0, OD600nm light absorption value tends towards stability, when pH liter
When up to 10.0, light absorption value dramatic decrease illustrates microorganism slow growth.Illustrate that the microorganism tenable environment range is wide, initially
For pH range at 7.0, growth performance is best.
(2) strategic point of making pottery Salmonella (Thauera sp.) CM-HZX2 optimum growth temperature
Culture medium after selection optimization, takes different cultivation temperatures respectively, 150r/min shaken cultivation, measures after 72h
Bacterial growth amount.Setting temperature is respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, at the same with the Salmonella (Thauera in distress that makes pottery is not added
Sp.) CM-HZX2 does blank, separated in time sample detection OD600nm value.
As shown in Figure 4, for microbial flocculant CM-HZX2 when temperature is 30~35 DEG C, growth light absorption value is maximum, works as culture
When condition is 40 DEG C, light absorption value slowly declines, this may be the growth that high temperature inhibits cell, so as to cause under OD 600nm value
Drop.
(3) strategic point of making pottery Salmonella (Thauera sp.) CM-HZX2 the most suitable growth revolving speed
Initial pH control is 7.0, and temperature is set as 30 DEG C, and revolving speed is successively set as 50r/min, 100r/min, 150r/
Min, 200r/min, 250r/min, shaken cultivation 72h under the conditions of 30 DEG C, while with Salmonella (Thauera sp.) in distress of making pottery is not added
CM-HZX2 does blank, separated in time sample detection OD 600nm value.
With the raising of shaking speed, oxygen-supplying amount is gradually increased, molten to obtain in the case of testing different shaking speeds
Solution oxygen acts on the influence of microorganism growth.As a result See Figure 5.As can be seen that as revolving speed increases in microculture condition,
OD600nm light absorption value is also becoming larger.In 150r/min, light absorption value is maximum, and revolving speed increases, and degradation rate tends to be steady.
Embodiment 3 is made pottery the salt-resistance of Salmonella in distress (Thauera sp.) CM-HZX2
In complicated industrial wastewater, salt content is higher, by changing salt content in culture medium, to investigate Salmonella bacterium in distress of making pottery
The saline-alkaline tolerance of strain.LB culture medium is prepared, switching volume is about 5%, initial pH 7, and at 30 DEG C, 150r/min shaking table is trained
It supports, separated in time section, separated in time sample detection OD 600nm value.
As a result See Figure 6, when concentration is 0~4%, with the increase of salinity, the OD 600nm of microorganism growth stablizes,
In 5% salinity, growth light absorption value decline illustrates that CM-HZX2 belongs to halotolerant strain.
Effect of the pottery luer bacteria strain of embodiment 4 to aqueous suspension ofkaolin
It takes a certain amount of bacterium solution to be transferred to LB culture medium, is 7 in initial pH, in 30 DEG C, 150r/min shaking table culture, 72h
Afterwards, it samples, measures the flocculating rate of the fermenation raw liquid in aqueous suspension ofkaolin, do blank, aqueous suspension ofkaolin flocculation with distilled water
It is 92.3% that rate, which is up to,.
Effect of the pottery luer bacteria strain of embodiment 5 to sanitary wastewater
It takes a certain amount of bacterium solution to be transferred to LB culture medium, is 7 in initial pH, in 30 DEG C, 150r/min shaking table culture, 72h
Afterwards, it samples, measures the flocculating rate of the fermenation raw liquid in sanitary wastewater, do blank with distilled water, waste water flocculating rate is up to
85.4%.
Embodiment 6 makes pottery luer bacteria strain to the flocculating rate for simulating industrial wastewater
It takes a certain amount of bacterium solution to be transferred to LB culture medium, is 7 in initial pH, in 30 DEG C, 150r/min shaking table culture, 72h
Afterwards, it samples, measures the flocculating rate of the fermenation raw liquid in waste water, do blank with distilled water, it is 80.4% that waste water flocculating rate, which is up to,.
The pottery luer bacteria strain of embodiment 7 is applied to the improvement of sanitary wastewater
By bacterial strain of the invention: using the mixture of polyvinyl alcohol and sodium alginate as carrier, and add active carbon, boric acid,
Calcium chloride etc., which embed, is prepared into microbe microsphere, each material composition and embedding method reference literature: rascal Buddhist nun, Cui Yingde,
The research of Yan Zhaoqiang, PVA- Alginate-Active Carbon Copolymer Hydrogel Oxygen Permeability, material Leader, 2008,22 (4).
Using certain plant area's sanitary wastewater as process object, its waste water is taken 30%, 60%, 100% micro- life to be added by volume
It is gradually tamed in object microballoon, the domestication time is 3 days, 30~35 DEG C of temperature, microbe microsphere is made to be applicable in wastewater environment.
By the microbe microsphere for being embedded with bacterium of the present invention tamed by volume 10% be added in advance it is constructed
In reactor, which has the function of aeration, water circulation and microballoon retention etc.；
Water quality is poor before administering, and has peculiar smell, transparency is low；After improvement, water quality is limpid, and transparency is high.
Claims (1)
1. application of the microbe microsphere in processing sanitary wastewater or industrial wastewater, which is characterized in that the microbe microsphere packet
The luer bacteria strain containing pottery；
The pottery luer bacteria strain is named as pottery strategic point Salmonella (Thauera sp.) CM-HZX2, and deposit number is CCTCC NO:
M2015689, the deposit date is in November, 2015；
The 16S rDNA sequence of described pottery strategic point Salmonella (Thauera sp.) CM-HZX2 is as shown in SEQ ID NO.1；
The biological property of described pottery strategic point Salmonella (Thauera sp.) CM-HZX2 are as follows: bacterium colony surface is round, wet, yellowish
Color, it is transparent, be easy to provoke, neat in edge, Gram's staining reaction is negative；
The cultural method of the Salmonella in distress of making pottery are as follows: after the pottery luer bacteria strain is activated, be inoculated into initial pH value be 6~9,
In the culture medium that temperature is 25~35 DEG C, the mass concentration of salt is 0~5%, cultivated under 150~200r/min revolving speed；
The application includes: then biochemical treatment to be carried out in waste water for after the pottery luer bacteria strain immobilization.
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| CN201610626905.9A CN106047768B (en) | 2016-08-02 | 2016-08-02 | A kind of pottery luer bacteria strain and its application |
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| CN106047768B true CN106047768B (en) | 2019-07-30 |
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| CN116855415A (en) * | 2023-07-12 | 2023-10-10 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Novel efficient flocculation denitrification bacterium and application thereof |
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