CN107129982A - A kind of preparation method and application of polyP bacteria trace charcoal - Google Patents
A kind of preparation method and application of polyP bacteria trace charcoal Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
- C02F2003/003—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms using activated carbon or the like
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
A kind of preparation method and application of polyP bacteria trace charcoal of the present invention, the charcoal for the load polyP bacteria that the present invention is provided can provide a good living environment for microorganism, and may also help in polyP bacteria improve resist high salinity and the ability of heavy metal, adsorption site increases simultaneously, and with suitable stability, the absorption property of heavy metal is greatly improved, product is nontoxic, environmentally friendly, the preparation method that the present invention is provided is simple, it is easy to industrialized production.
Description
Technical field
The present invention relates to a kind of preparation method of polyP bacteria trace charcoal, more particularly to a kind of polyP bacteria trace charcoal
Preparation method and application.
Background technology
There are the biomass such as substantial amounts of sawdust, straw in China by waste is burned every year, and this can not only be caused environment pollution
Harm, can also cause the waste and loss of bioenergy.How fully and reasonably utilize this kind of " waste material ", make its change give up into
Treasured, this is to need people's urgent problem to be solved.By pyrolysis, modified obtained charcoal there is certain Adsorption of Heavy Metals to make
With can reduce migration, the biological effectiveness of environment heavy metal, be widely studied, however, single charcoal is due to table itself
Face adsorption site is very limited, and its absorption property and stability are very limited.Therefore, what research and development were new has high suction
The stable and cheap practical adsorbent of attached capacity, passivation properties, is the key of its further development and application, with important meaning
Justice.
It is a common quasi-microorganism in slag/soil to gather Phosphorus strain, and it can include Cr, U as environment support sticking
Pollutant Deng including, or effect precipitated etc. by mineralising change Cr, Pb form and adsorb in fixation, and then reduction waste water
The content of heavy metal or the migration of heavy metal in soil, biological effectiveness and toxicity.In order that polyP bacteria is fixed on charcoal
Surface, increase polyP bacteria attachment adsorption site position and stability, under the protection of carrier improve cell heavy metal it is resistance to
By property, increase the biomass in unit volume, the effect of collaboration charcoal improves the adsorption efficiency of heavy metal.
The content of the invention
Present invention aims at the technical problem existed for prior art, a kind of high-adsorption-capacity, property are developed stable
With the cheap practical immobilized microorganism trace charcoal available for adsorption treatment heavy metal ion.
To achieve the above object, the technical scheme that the present invention is provided comprises the following steps：
(1) 5-10g sawdusts are taken, anaerobism isothermal pyrolysis 4h is carried out at a temperature of 200-700 DEG C；Completely room is cooled to after charing
Temperature is taken out, and obtains charcoal, and charcoal is mixed and ground, and is crossed 100 mesh sieves, is saved backup in drier；
(2) take 10g activated sludge to add in 90ml sterilized waters to mix, be stored at room temperature 10min, take supernatant dilute by 10 times
It is respectively 10 that interpretation of the law, which is made into concentration,-1To 10-66 kinds of bacteria suspensions, the bacterium solution even spread of 0.2ml various concentrations is taken respectively
Onto the rich phosphorus flat board of solid, in 30 DEG C of incubator culture 24h, in the gradient plate for obvious single bacterium colony occur, picking single bacterium colony
It is transferred to the single bacterium colony form being purified to repeatedly on rich phosphorus culture medium on flat board consistent, obtains just pure bacterial strain, be connected to LB solid cultures
On base inclined-plane, 4 DEG C save backup；
(3) by pure bacterial strain access domestication culture medium triangular flask at the beginning of a part, bottleneck is sealed with eight layers of gauze, is placed in constant temperature
Culture shaking table carries out aerobic culture 24h, the micro- aerobic culture bar of sem observation under the conditions of being 150r/min as 30 DEG C, rotating speed using temperature
Part hypothallus accumulates the situation of polyphosphate particle；In addition, pure bacterial strain at the beginning of another is accessed in another domestication culture medium triangular flask, with
Nitrogen will be filled with bottle afterwards to be made to be in anaerobic state in bottle, and aerobic culture 24h, micro- sem observation anaerobism are carried out with rubber stopper seal
The situation of accumulation PHB particles in condition of culture hypothallus；The polyphosphate particle and anaerobism accumulated respectively to thalline in aerobic culture is trained
Support the tired PHB particles of condition hypothallus inner product to be dyed, it is primary dcreening operation to be chosen at the thalline being all positive in two kinds of colouring methods
Bacterium；
(4) primary dcreening operation bacterial strain is accessed in poly- phosphorus nutrient solution, is 150r/min shaking table shaken cultivation 12h in 30 DEG C, rotating speed, so
- 30 DEG C of refrigerated centrifuge 10min under 10000r/min rotating speeds, take supernatant to dilute afterwards, with molybdenum-antimony anti-spectrophotometric method,
Its absorbance is measured at 700nm, the clearance of phosphorus is calculated, the bacterial strain for taking the poly- phosphorus rate of thalline to be more than 60% is secondary screening bacterium；
(5) step 1) in obtained phosphorus base charcoal and secondary screening bacterium according to mass ratio 20:1 ratio, is separately added into fixation
Change in culture medium, Immobilized culture base includes：Sucrose 10g/L, beef extract 6g/L, dusty yeast 1.5g/L, pH7.0, by immobilization
Culture medium concussion and cultivate 18h in 30 DEG C of temperature, 160r/min constant-temperature table, then goes out to adsorb with the screen filtration of 30 mesh
The phosphorus base charcoal of thalline, i.e. polyP bacteria trace charcoal.
It is preferred that, charcoal anaerobism isothermal pyrolysis temperature is prepared for 400-500 DEG C.
It is preferred that, described rich phosphorus culture medium includes：Beef extract 3g/L, peptone 10g/L, sodium chloride 5g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.2g/L, agar 18g/L, rich phosphorus medium pH are 7.0.
It is preferred that, described domestication culture medium includes：Sodium acetate 0.34g/L, ammonium chloride 0.076g/L, magnesium sulfate 0.02g/
L, calcium chloride 0.01g/L, potassium dihydrogen phosphate 0.09g/L, sodium acid carbonate 0.3g/L, micro-mixed liquor 1ml, tame culture medium
pH7.5。
It is preferred that, described micro-mixed liquor includes：Ethanedioic acid triethylammonium tetrakis 10g/L, ferrous sulfate 1.54g/L, boron
Sour 0.15g/L, cupric sulfate pentahydrate 0.03g/L, manganese chloride 0.12g/L, KI 0.18g/L, sodium molybdate 0.06g/L, seven water sulphur
Sour zinc 0.12g/L, six hydration cobaltous dichloride 0.15g/L.
It is preferred that, described poly- phosphorus nutrient solution includes：Beef extract 0.22g/L, sodium acetate 0.5g/L, magnesium sulfate 0.4g/L,
Ferrous sulfate 0.002g/L, copper sulphate 0.08g/L, ammonium sulfate 0.2g/L, dipotassium hydrogen phosphate 0.0147g/L, poly- phosphorus nutrient solution
pH7.0。
Present invention also offers a kind of application of polyP bacteria trace charcoal, described polyP bacteria trace charcoal is by above-mentioned
The preparation method of the biological carbon adsorbent of polyP bacteria trace a kind of be made, it is applied to the processing of heavy metal wastewater thereby.
It is preferred that, polyP bacteria trace charcoal consumption in heavy metal wastewater thereby is 0.3~0.6g/L.
It is preferred that, polyP bacteria trace charcoal reaction condition in heavy metal wastewater thereby is：Heavy metal wastewater thereby pH value 5~7, instead
It is 3~6 hours between seasonable.
Compared with prior art, the beneficial effects of the invention are as follows：
Biomass carbon disclosed in this invention is prepared in the range of 400-500 DEG C of preferable temperature, prepared by reduction to greatest extent
The loss of process organic carbon, and the stability of formed height aromatization organic compound is improved as far as possible；Then will culture
Pseudomonad to exponential phase is inoculated into fermentation tank, and adds charcoal, and the processing of being fixed of polyP bacteria is obtained
PolyP bacteria trace biology carbon adsorbent.
The charcoal of the load polyP bacteria prepared using the inventive method has especially excellent for heavy metal containing wastewater treatment
Good effect, by taking heavy metal Cr and metal U ions as an example, the charcoal for loading polyP bacteria is added to containing in Cr and U solution, passed through
PolyP bacteria acts synergistically with charcoal, and absorption quickly reaches balance, and it can be such that chromium, metal uranium content in waste water declines respectively
80.2-82.1% and 78.9-82.7%.
The present invention uses primary raw material sawdust wide material sources, and preparing charcoal makes it turn waste into wealth, other such as dense nitre
Acid, calcium phosphate is conventional chemical products；PolyP bacteria is common in the excess sludge of city domestic sewage processing, is separated, and is extracted
It is convenient.
Compared with existing charcoal or class charcoal adsorber technologies, the charcoal for the load polyP bacteria that the present invention is provided can
High salinity and heavy metal are resisted to provide a good living environment for microorganism, and may also help in polyP bacteria to improve
Ability, while adsorption site increases, and with suitable stability, the absorption property of heavy metal is greatly improved；Product without
Poison, environmentally friendly, the preparation method that the present invention is provided is simple, it is easy to industrialized production.
Brief description of the drawings
The invention will be further described below in conjunction with the accompanying drawings：
The scanning electron microscope (SEM) photograph of Fig. 1 polyP bacteria trace charcoals；
The magnified sweep electron microscope of Fig. 2 polyP bacteria trace charcoals；
The energy spectrum diagram of Fig. 3 polyP bacteria trace charcoals；
The scanning electron microscope (SEM) photograph of the common charcoals of Fig. 4；
The magnified sweep electron microscope of the common charcoals of Fig. 5；
The energy spectrum diagram of the common charcoals of Fig. 6；
The examination of infrared spectrum figure of the common charcoals of Fig. 7 and polyP bacteria trace charcoal.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.
Embodiment 1：
(1) progress anaerobism isothermal pyrolysis 4h at a temperature of 5~10g sawdusts, 200-700 DEG C is taken；Completely room is cooled to after charing
Temperature is taken out, and obtains charcoal, and charcoal is mixed and ground, and is crossed 100 mesh sieves, is saved backup in drier；
(2) take 10g activated sludge to add in 90ml sterilized waters to mix, be stored at room temperature 10min, take supernatant dilute by 10 times
It is respectively 10 that interpretation of the law, which is made into concentration,-1To 10-66 kinds of bacteria suspensions, the bacterium solution even spread of 0.2ml various concentrations is taken respectively
Onto the rich phosphorus flat board of solid, in 30 DEG C of incubator culture 24h, in the gradient plate for obvious single bacterium colony occur, picking single bacterium colony
It is transferred to the single bacterium colony form being purified to repeatedly on rich phosphorus culture medium on flat board consistent, obtains just pure bacterial strain, be connected to LB solid cultures
On base inclined-plane, 4 DEG C save backup；
(3) by pure bacterial strain access domestication culture medium triangular flask at the beginning of a part, bottleneck is sealed with eight layers of gauze, is placed in constant temperature
Culture shaking table carries out aerobic culture 24h, the micro- aerobic culture bar of sem observation under the conditions of being 150r/min as 30 DEG C, rotating speed using temperature
Part hypothallus accumulates the situation of polyphosphate particle；In addition, pure bacterial strain at the beginning of another part is accessed in another domestication culture medium triangular flask,
Nitrogen will be then filled with bottle to be made to be in anaerobic state in bottle, aerobic culture 24h is carried out with rubber stopper seal, micro- sem observation is detested
The situation of accumulation PHB particles in oxygen condition of culture hypothallus；The polyphosphate particle and anaerobism accumulated respectively to thalline in aerobic culture
The PHB particles of accumulation are dyed in condition of culture hypothallus, and it is first to be chosen at the thalline being all positive in two kinds of colouring methods
Sieve bacterium；
Polyphosphate particle Sudan Black staining method：
(1) dyeing liquor
Solution A：The ethanol 2ml of volume fraction 95%, toluidine blue 0.15g, glacial acetic acid 1ml, peacock green 0.2g, distilled water
100ml.First dyestuff is dissolved in ethanol, the glacial acetic acid and distilled water mixed in advance is added thereto, filters standby after placing 24h
With；
Second liquid：Iodine 2g, KI 3g, distilled water 100ml.
(2) staining procedure
Sterilized water is dripped on slide, a small amount of bacterium colony is dipped in sterilized water with collarium is connect, and is air-dried and is fixed；Use solution A
5min is contaminated, solution A of inclining washes away solution A with second liquid, and dye 1min, with being blotted after distilled water flushing；There is different dye in polyP bacteria body
Coloured particles, the positive has atrament, and other parts are bottle green or light green color.
PHB granules stains
(1) dyeing liquor
Sudan black 3g/L：Sudan black 0.3g, the ethanol 100ml of volume fraction 70%, mixing forced oscillation is stood overnight standby
With.
Dimethylbenzene：Decolourant Huang red aqueous solution 50g/L：Redye liquid
(2) staining procedure
Routinely smear is made in dyeing, and 5min is dyed with solution A, and solution A of inclining washes away solution A with second liquid, and contaminate 1min, water
Wash, blot, microscopy, after dyeing, the positive can be changed into black-and-blue to PHB, and other parts are red.
(4) primary dcreening operation bacterial strain is respectively connected in the triangular flask equipped with the poly- phosphorus nutrient solutions of 50mL, is 150r/ in 30 DEG C, rotating speed
Min shaking table shaken cultivation 12h, then centrifuge 10min under 10000r/min rotating speeds, pour out supernatant, pipette 2mL and be diluted to
50mL, is compareed with the poly- phosphorus nutrient solution for not being inoculated with bacterium, and with molybdenum-antimony anti-spectrophotometric method, its absorbance is measured at 700nm, is led to
Cross standard curve and check in phosphorus concentration, calculate the clearance of phosphorus, the bacterial strain for taking the poly- phosphorus rate of thalline to be more than 60% is secondary screening bacterium；
(5) 4g steps 1 are weighed) in obtained phosphorus base charcoal, while configuring Immobilized culture base, Immobilized culture Ji Bao
Include：Sucrose 10g/L, beef extract 6g/L, dusty yeast 1.5g/L, pH7.0；Answering after activation is accessed by 5% mass concentration inoculum concentration
Bacterium is sieved, load weighted phosphorus base charcoal is added, 30 DEG C are put into, concussion and cultivate 18h in 160r/min constant-temperature table, then with 30
Purpose screen filtration goes out to have adsorbed the phosphorus base charcoal of thalline, i.e. polyP bacteria trace charcoal.
PolyP bacteria trace charcoal outward appearance made from above-mentioned steps is in black, and specific surface area is in 25.42-56.27m2/ g it
Between, be placed under ESEM and observe, its structure as Figure 1-3, by with SEM-EDS couples of Fig. 4-6 (common charcoal)
Than the P elements that can be seen that the biological carbon surface 0.16% of polyP bacteria trace, illustrate that a certain proportion of phosphorous bacterium is contained on its surface
Strain, i.e. polyP bacteria.
Embodiment 2
The polyP bacteria trace charcoal of the present invention is applied to the Cr ions in processing waste water, comprises the following steps：Take 100mL
Initial concentration is 10~100mg/L Cr6+Solution, the pH value of regulation solution is 6.0, adds polyP bacteria print made from embodiment 1
Mark biology carbon adsorbent, the consumption of the adsorbent is 0.25g/L, and adsorption reaction 3 hours is carried out in 30 DEG C of constant temperature oscillators, then will
The adsorbent is separated from waste water, and remaining Cr in waste water is determined with AAS6+Content, the adsorbance result such as institute of table 1 of calculating
Show：
Table 1：PolyP bacteria trace charcoal is to different Cr6+The adsorbance of initial concentration waste water
As shown in Table 1, the adsorbent has 8.75mg/g adsorbance under conditions of initial concentration is 10.01mg/L,
And increase with the increase of Cr6+ initial concentrations, to certain value after 25.44mg/g basically reach stabilization.
Embodiment 3
The polyP bacteria trace charcoal of the present invention is used to handle the radioactive metal UO in waste water2+Ion, including following step
Suddenly：Take the UO that 100mL initial concentrations are 10~100mg/L2+Solution, the pH value of regulation solution is 6.0, adds embodiment 1 and is made
The biological carbon adsorbent of polyP bacteria trace, the consumption of the adsorbent is 0.25g/L, and adsorption reactions are carried out in 30 DEG C of constant temperature oscillators
3 hours, then the adsorbent is separated from waste water, determine remaining UO in waste water with AAS2+Content, the adsorbance knot of calculating
Fruit is as shown in table 1：
Table 2：PolyP bacteria trace charcoal is to different Cr6+The adsorbance of initial concentration waste water
As shown in Table 2, the adsorbent has 17.13mg/g adsorbance under conditions of initial concentration is 10.03mg/L,
And with UO2+The increase of initial concentration and increase, to certain value after 26.52mg/g basically reach stabilization.
Described above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement should be regarded as the guarantor of the present invention
Protect scope.
Claims (10)
1. a kind of preparation method of polyP bacteria trace charcoal, it is characterised in that comprise the following steps：
(1) 5-10g sawdusts are taken, anaerobism isothermal pyrolysis 4h is carried out at a temperature of 200-700 DEG C；Room temperature is cooled to after charing completely to take
Go out, obtain charcoal, charcoal is mixed and ground, cross 100 mesh sieves, saved backup in drier；
(2) take 10g activated sludge to add in 90ml sterilized waters to mix, be stored at room temperature 10min, take supernatant doubling dilution, it is different
Gradient doubling dilution liquid, which is utilized respectively, is down flat plate cultural method culture, in the gradient plate for obvious single bacterium colony occur, separates pure
Change to the single bacterium colony form on flat board unanimously, obtain just pure bacterial strain, be connected in LB culture medium slants, 4 DEG C save backup；
(3) it is 150r/min conditions by 30 DEG C, rotating speed of temperature in pure bacterial strain access domestication culture medium triangular flask at the beginning of taking a part
The aerobic culture 24h of lower progress constant temperature；In pure another domestication culture medium triangular flask of bacterial strain access at the beginning of taking another part, it will be filled with bottle
Nitrogen carries out Anaerobic culturel 24h；In the polyphosphate particle and anaerobic culture conditions hypothallus accumulated respectively to thalline in aerobic culture
Accumulation PHB particles are dyed, and it is primary dcreening operation bacterium to be chosen at the thalline being all positive in two kinds of colouring methods；
(4) primary dcreening operation bacterial strain is accessed in poly- phosphorus nutrient solution, is 150r/min shaking table shaken cultivations 12h, Ran Hou in 30 DEG C, rotating speed
- 30 DEG C of refrigerated centrifuge 10min, take supernatant to dilute under 10000r/min rotating speeds, with molybdenum-antimony anti-spectrophotometric method, at 700nm
Its absorbance is measured, the clearance of phosphorus is calculated, the bacterial strain for taking the poly- phosphorus rate of thalline to be more than 60% is secondary screening bacterium；
(5) step 1) in obtained phosphorus base charcoal and secondary screening bacterium according to mass ratio 20:1 ratio, is separately added into immobilization training
Support in base, the concussion and cultivate 18h in 30 DEG C of temperature, 160r/min constant-temperature table, then go out absorption with the screen filtration of 30 mesh
The phosphorus base charcoal of good thalline, i.e. polyP bacteria trace charcoal.
2. the preparation method of a kind of polyP bacteria trace charcoal according to claim 1, it is characterised in that prepare charcoal
Anaerobism isothermal pyrolysis temperature is 400-500 DEG C.
3. a kind of preparation method of polyP bacteria trace charcoal according to claim 1, it is characterised in that described dilution
Being down flat plate cultural method is：It is respectively 10 to take supernatant to be made into concentration by 10 times of dilution methods-1To 10-66 kinds of bacterium hang
Liquid, takes the bacterium solution even spreads of 0.2ml various concentrations to the rich phosphorus flat board of solid, in 30 DEG C of incubator culture 24h respectively.
4. a kind of preparation method of polyP bacteria trace charcoal according to claim 1, it is characterised in that described rich phosphorus
Culture medium includes beef extract 3g/L, peptone 10g/L, sodium chloride 5g/L, potassium dihydrogen phosphate 0.2g/L, agar 18g/L, rich phosphorus training
It is 7.0 to support base pH.
5. a kind of preparation method of polyP bacteria trace charcoal according to claim 1, it is characterised in that described domestication
Culture medium includes sodium acetate 0.34g/L, ammonium chloride 0.076g/L, magnesium sulfate 0.02g/L, calcium chloride 0.01g/L, potassium dihydrogen phosphate
0.09g/L, sodium acid carbonate 0.3g/L, micro-mixed liquor 1ml, tame medium pH 7.5.
6. the preparation method of a kind of polyP bacteria trace charcoal according to claim 5, it is characterised in that described is micro
Element mixed liquor includes：Ethanedioic acid triethylammonium tetrakis 10g/L, ferrous sulfate 1.54g/L, boric acid 0.15g/L, cupric sulfate pentahydrate 0.03g/
L, manganese chloride 0.12g/L, KI 0.18g/L, sodium molybdate 0.06g/L, white vitriol 0.12g/L, six hydration cobaltous dichlorides
0.15g/L。
7. a kind of preparation method of polyP bacteria trace charcoal according to claim 1, it is characterised in that described poly- phosphorus
Nutrient solution includes：Beef extract 0.22g/L, sodium acetate 0.5g/L, magnesium sulfate 0.4g/L, ferrous sulfate 0.002g/L, copper sulphate
0.08g/L, ammonium sulfate 0.2g/L, dipotassium hydrogen phosphate 0.0147g/L, poly- phosphorus nutrient solution pH7.0.
8. the application of the biological carbon adsorbent of a kind of polyP bacteria trace, it is characterised in that described polyP bacteria trace charcoal is by upper
The preparation method for stating a kind of polyP bacteria trace charcoal described in 1-7 is made, and it is applied to the processing of heavy metal wastewater thereby.
9. a kind of application of the biological carbon adsorbent of polyP bacteria trace according to claim 8, it is characterised in that the poly- phosphorus
Bacterium trace charcoal consumption in heavy metal wastewater thereby is 0.3~0.6g/L.
10. the application of the biological carbon adsorbent of a kind of polyP bacteria trace according to claim 8, it is characterised in that described poly-
Phosphorus bacterium trace charcoal reaction condition in heavy metal wastewater thereby is：Heavy metal wastewater thereby pH value 5~7, the reaction time is 3~6h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113213579A (en) * | 2021-05-25 | 2021-08-06 | 贵州省材料产业技术研究院 | Application of photocatalytic biochar composite material in catalytic degradation of printing and dyeing wastewater |
| CN113818152A (en) * | 2021-09-26 | 2021-12-21 | 厦门大学 | Preparation method and application of biomass carbon nanofiber membrane loaded with microorganisms |
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