CN107058207A - A kind of rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading - Google Patents

A kind of rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading Download PDF

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CN107058207A
CN107058207A CN201710251089.2A CN201710251089A CN107058207A CN 107058207 A CN107058207 A CN 107058207A CN 201710251089 A CN201710251089 A CN 201710251089A CN 107058207 A CN107058207 A CN 107058207A
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bisphenols
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周集体
魏浩
吕红
王竞
张倩
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Dalian University of Technology
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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract

The invention belongs to technical field of environmental microorganism, it is related to the rejuvenation of bacterial strain, is related specifically to a kind of rejuvenation method for the heterotrophic nitrification aerobic denitrifying bacterium for handling bisphenols nitric wastewater.Cultivated using the culture medium containing common carbon source, it is gradually added into the nitrogenous water distribution of bisphenols and improves its proportioning, so that microorganism is by an incremental domestication process and gradually recovers its adaptability to bis-phenol pollutant, the heterotrophic nitrification aerobic denitrifying bacterial strain using bis-phenol pollutant as carbon source is finally obtained.This method cultivates the method for carrying out rejuvenation relative to the direct culture medium with containing common carbon source or the nitrogenous water distribution of bisphenols repeatedly, and rejuvenation has higher success rate, and this method is operationally simple and easy to apply, time-consuming less.And the degraded situation by the use of bis-phenol pollutant, ammonia nitrogen removal frank and its conversion ratio of gaseous nitrogen etc. is converted into as the index for evaluating rejuvenation bacterial strain performance, it is relatively comprehensive.

Description

A kind of rejuvenation of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading Method
Technical field
The invention belongs to field of environment microorganism, it is related to the rejuvenation of bacterial strain, is related specifically to a kind of processing bisphenols and contains The rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium of nitrogen sewage.
Background technology
Traditional ammonia-nitrogen sewage processing needs the nitrification by autotrophy and two stages ability of denitrification of heterotrophism It can complete.Because the growth rate of Autotrophic nitrification bacterium causes nitrification efficiency relatively low slowly, so as to considerably increase processing procedure Time-consuming, the stability of whole sewage disposal process denitrification effect is also easily affected.And the nitrification and denitrification stage needs Separately carry out adding energy consumption, while floor space is larger, investment and operating cost are larger.
The discovery and application of heterotrophic nitrification aerobic denitrifying bacteria largely solve above mentioned problem so that aerobic Under the conditions of nitrification and denitrification be possibly realized in same reactor.It is different when dirty organic pollutants have more Type bacterium is supported because comparatively fast, so as to easily become dominant bacteria, particularly many heterotrophic nitrifications are good using organic matter multiplication rate Oxygen denitrifying bacteria can be carbon source using hardly degraded organic substance, and this goes for dirty Organic substance in water particularly hardly degraded organic substance Except with denitrogenation important in inhibiting.
Usual heterotrophic nitrification aerobic denitrifying bacterium passes through the Effec-tive Function of certain time, occurs specific due to iterating Activity is degenerated, or function reduction phenomenon occurs, it is necessary to rejuvenation be carried out to it, to maintain its denitrogenation because culture presevation is improper The stability of energy.The method that existing rejuvenation of spawn technology is typically cultivated repeatedly using bacterium culture medium, but it is generally multiple Strengthen less effective and time-consuming longer.Therefore propose that the efficient rejuvenation method of heterotrophic nitrification aerobic denitrifying bacterium has important meaning Justice.The invention provides a kind of rejuvenation side of good, the quick and simple and easy to apply heterotrophic nitrification aerobic denitrifying bacterium of effect of rejuvenation Method.
The content of the invention
For deficiency present in existing rejuvenation technique, it is an object of the invention to provide one kind degraded nitrogenous dirt of bisphenols The rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium of water, to provide lasting microorganism for the processing of bisphenols nitric wastewater Source.
Technical scheme：
A kind of rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading, step is as follows：
(1) domestication culture is carried out to the strain for needing rejuvenation：
(1.1) fluid nutrient medium I is prepared, the concentration proportioning of various materials is：(NH4)2SO40.1-0.5g/L, citric acid Sodium 1-3g/L, sodium acetate 1-3g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O 0.2-0.8g/L、 CaCl20.02-0.1g/L, pH are adjusted to 6.8-7.2；
(1.2) the nitrogenous water distribution of bisphenols is prepared, the concentration proportioning of various materials is：(NH4)2SO4It is 0.01-0.02g/L, double Aldehydes matter 0.2-0.5g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O0.2-0.8g/L、CaCl2 0.02-0.1g/L, pH are adjusted to 6.8-7.2；
The nitrogenous water distribution of the fluid nutrient medium I and bisphenols of preparation is subjected to sterilization treatment；
(1.3) three class I liquid I culture mediums are aseptically prepared：
The first kind is the fluid nutrient medium I of 100% volume；
Equations of The Second Kind includes multiple fluid nutrient mediums, each nitrogenous by the fluid nutrient medium I and bisphenols of different volumes ratio Water distribution is made into, and the volume ratio shared by fluid nutrient medium I is prepared by the order successively decreased, fluid nutrient medium I volume ratio Scope is 90%~10%；Volume ratio shared by the nitrogenous water distribution of bisphenols is prepared by incremental order, the nitrogenous water distribution of bisphenols Volume ratio scope be 10%~90%；
3rd class is the nitrogenous water distribution of bisphenols of 100% volume；
The three class I liquid I culture mediums prepared are subjected to sterilization treatment；
(1.4) strain for needing rejuvenation is carried out into progressively domestication to cultivate, first by first kind fluid nutrient medium, domestication training Support 1-2d；The incremental order of volume ratio again as shared by the nitrogenous water distribution of bisphenols in matched somebody with somebody fluid nutrient medium changes Equations of The Second Kind liquid Body culture medium, often changes once domestication culture 1-2d；Finally change the 3rd class I liquid I culture medium, domestication culture 1-2d；
What domestication was cultivated concretely comprises the following steps：Aseptically, the heterotrophism of rejuvenation will be needed with phosphate buffer first Nitrification aerobic denitrification strain is cleaned and centrifuged 2-3 times, removes supernatant, is subsequently added into phosphate buffer and is carried out concussion weight It is outstanding, uniform bacterium solution is obtained, bacterium solution is pipetted and is seeded in the fluid nutrient medium of step (1.3) preparation so that the concentration of initial strain For OD600=0.1-0.15, finally by fluid nutrient medium be placed in it is sterile, 30-35 DEG C, tamed under conditions of 100-150rpm Culture；Wherein, the concentration proportioning of each material is in phosphate buffer：K2HPO4·2H2O 5-6g/L、KH2PO42-3g/L, will Phosphate buffer pH is adjusted to 6.8-7.2；Centrifugal condition is：8000-10000×g、5min、4℃；OD600For in 600nm ripples The bacterial concentration measured under length；
(2) bacterium after domestication culture is isolated and purified：
Solid medium is prepared, the concentration proportioning of various materials is：(NH4)2SO40.1-0.5g/L, sodium citrate 1-3g/ L, sodium acetate 1-3g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O0.2-0.8g/L、CaCl2 0.02-0.1g/L, agar 20g/L, pH are adjusted to 6.8-7.2；
The microbionation after culture will be tamed with plate streak to the solid medium that sterilizes, flat board is inverted in 30-35 Culture 2-3d is to obtain single bacterium colony in DEG C constant incubator；
(3) primary dcreening operation is carried out to efficient bacterium：
A. the single bacterium colony in picking flat board is seeded in first kind fluid nutrient medium respectively, by the first kind liquid after inoculation Body culture medium is placed in 30-35 DEG C, isothermal vibration culture 1-2d under the conditions of 100-150rpm, and ammonia nitrogen is measured by sampling and total nitrogen is removed Rate；
B. ammonia nitrogen and total nitrogen removal level to measure reaches the bacterial strain of the normal removal level more than 80% of strain, with OD600 It is seeded to respectively in the 3rd class I liquid I culture medium for 0.1-0.15 inoculum concentration, the 3rd class I liquid I culture medium after inoculation is placed in 30-35 DEG C, isothermal vibration culture 2-3d under the conditions of 100-150rpm, are measured by sampling bis-phenol pollutant, ammonia nitrogen and total nitrogen and remove Rate；
Repeat step B, until the bis-phenol pollutant, ammonia nitrogen and total nitrogen of measure remove the normal removal level before degeneration that reaches And keep stable.
(4) nitrogen balance analysis is carried out to efficient bacterium.By the bacterial strain obtained in step (3) with OD600For connecing for 0.1-0.15 The amount of kind is seeded in the nitrogenous water distribution of bisphenols, and the gaseous mixture (volume ratio 3/7) that access is made up of oxygen and helium is aerated, Reaction system is subjected to encapsulation process after 10-20min to be aerated, the static gas wave refrigerator 2-3d in 30-35 DEG C of incubator.Produce Gas is determined with gas chromatography (GC), obtains N2And N2O concentration；Determine liquid portion total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrous The concentration of state nitrogen, pellet fraction is handled with freeze-drying, the concentration of organic nitrogen in somatic cells is determined, by determining nitrogen Amount, which is calculated, determines that bacterial strain, to the metabolic characteristic of ammonia nitrogen, obtains the heterotrophic nitrification aerobic denitrifying of efficient degradation bisphenols nitric wastewater Bacterial strain.
The effect and benefit of the present invention：The invention provides a kind of efficient, simple and easy to apply heterotrophic nitrification aerobic denitrifying The rejuvenation method of bacterium.Cultivated using the culture medium containing common carbon source, be gradually added into the nitrogenous water distribution of bisphenols and improve it and match somebody with somebody Than so that microorganism is by an incremental domestication process and gradually recovers its adaptability to bis-phenol pollutant, Finally obtain the heterotrophic nitrification aerobic denitrifying bacterial strain using bis-phenol pollutant as carbon source.This method is relative to directly with containing common The method for carrying out rejuvenation is cultivated in the nitrogenous water distribution of culture medium or bisphenols of carbon source repeatedly, and rejuvenation has higher success rate, and in behaviour It is simple and easy to apply on work, take less.And using the degraded situation of bis-phenol pollutant, ammonia nitrogen removal frank and its it is converted into gaseous state Conversion ratio of nitrogen etc. is relatively more comprehensive as the index for evaluating rejuvenation bacterial strain performance.
Embodiment
Normal HS-2 strains are that the heterotrophic nitrification aerobic denitrifying with the nitrogenous water distribution function of efficient degradation Bisphenol F is thin Bacterium, the HS-2 bacterial strains that present embodiment has been degenerated to function carry out rejuvenation, and step is as follows：
(1) domestication culture is carried out to the HS-2 strains for needing rejuvenation：
(1.1) fluid nutrient medium I is prepared, the concentration proportioning of various materials is：(NH4)2SO40.3g/L, sodium citrate 2g/ L, sodium acetate 2g/L, K2HPO4·2H2O 5.5g/L、KH2PO4 2.6g/L、MgSO4·7H2O0.2g/L、CaCl20.02g/L, PH is adjusted to 7.0；
(1.2) the nitrogenous water distribution of Bisphenol F is prepared, the concentration proportioning of various materials is：(NH4)2SO40.012g/L, Bisphenol F 0.3g/L、K2HPO4·2H2O 5.5g/L、KH2PO4 2.6g/L、MgSO4·7H2O 0.2g/L、CaCl20.02g/L, pH are adjusted To 7.0；
The nitrogenous water distribution of the fluid nutrient medium I and Bisphenol F of preparation is subjected to sterilization treatment；
(1.3) three class I liquid I culture mediums are aseptically prepared：
The first kind is the fluid nutrient medium I of 100% volume；
Equations of The Second Kind includes multiple fluid nutrient mediums, each nitrogenous is matched somebody with somebody by the fluid nutrient medium I and Bisphenol F of different volumes ratio Water is made into, and the volume ratio shared by fluid nutrient medium I is prepared by the order successively decreased, fluid nutrient medium I volume ratio model Enclose for 80%, 60%, 40%, 20%；Volume ratio shared by the nitrogenous water distribution of Bisphenol F is prepared by incremental order, and Bisphenol F is nitrogenous The volume ratio scope of water distribution is 20%, 40%, 60%, 80%；
3rd class is the nitrogenous water distribution of Bisphenol F of 100% volume；
The three class I liquid I culture mediums prepared are subjected to sterilization treatment；
(1.4) strain for needing rejuvenation is carried out into progressively domestication to cultivate, first by first kind fluid nutrient medium, domestication training Support 1d；Again by the culture medium of the fluid nutrient medium I of 80% volume and the nitrogenous water distribution composition of the sterilizing Bisphenol F of 20% volume, 60% The culture medium of the fluid nutrient medium I of volume and the nitrogenous water distribution composition of the sterilizing Bisphenol F of 40% volume, the Liquid Culture of 40% volume Culture medium, the fluid nutrient medium I of 20% volume and 80% volume of the nitrogenous water distribution composition of sterilizing Bisphenol F of base I and 60% volume The order of culture medium of sterilizing Bisphenol F nitrogenous water distribution composition change Equations of The Second Kind fluid nutrient medium, often change once domestication culture 1d；Finally change the 3rd class I liquid I culture medium, domestication culture 1d；
What domestication was cultivated concretely comprises the following steps：Aseptically, the heterotrophism of rejuvenation will be needed with phosphate buffer first Nitrification aerobic denitrification strain is cleaned and centrifuged 2 times, removes supernatant, is subsequently added into phosphate buffer and is carried out concussion resuspension, Uniform bacterium solution is obtained, bacterium solution is pipetted and is seeded in the fluid nutrient medium of step (1.3) preparation so that initially the concentration of strain is OD600=0.1, finally by fluid nutrient medium be placed in it is sterile, 35 DEG C, domestication culture is carried out under conditions of 150rpm；Wherein, phosphoric acid The concentration proportioning of each material is in salt buffer：K2HPO4·2H2O 5.5g/L、KH2PO42.6g/L, by phosphate buffer pH Adjust to 7.0；Centrifugal condition is：10000×g、5min、4℃；OD600For the bacterial concentration measured under 600nm wavelength；
(2) bacterium after domestication culture is isolated and purified：
Solid medium is prepared, the concentration proportioning of various materials is：(NH4)2SO40.3g/L, sodium citrate 2g/L, acetic acid Sodium 2g/L, K2HPO4·2H2O 5.5g/L、KH2PO4 2.6g/L、MgSO4·7H2O 0.2g/L、CaCl20.02g/L, agar 20g/L, pH are adjusted to 7.0；
The microbionation after culture will be tamed with plate streak to the solid medium that sterilizes, flat board is inverted in 35 DEG C of perseverances 2d is cultivated in warm incubator to obtain single bacterium colony；
(3) primary dcreening operation is carried out to efficient bacterium：
A. totally 12 single bacterium colonies are seeded in the first kind fluid nutrient medium HAW1-12 in picking flat board respectively, will be inoculated with First kind fluid nutrient medium afterwards is placed in 35 DEG C, isothermal vibration culture 1d under the conditions of 150rpm, and ammonia nitrogen is measured by sampling and total nitrogen is gone Except rate；
B. ammonia nitrogen and total nitrogen removal level to measure reach strain normally the bacterial strain HAW2-4 of removal level more than 80%, HAW6, HAW8-10, with OD600It is seeded to respectively in the 3rd class I liquid I culture medium for 0.1 inoculum concentration, by the 3rd class after inoculation Fluid nutrient medium is placed in 35 DEG C, isothermal vibration culture 2d under the conditions of 150rpm, and Bisphenol F, ammonia nitrogen and nitrogen removal rate is measured by sampling；
Repeat step B, respectively reaches 99%, 96% and 90% and protects until filtering out Bisphenol F, ammonia nitrogen and nitrogen removal rate Two bacterial strains HAW3, HAW6 for keeping steady fixed.
(4) nitrogen balance analysis is carried out to efficient bacterium.By the bacterial strain HAW3 and HAW6 that are obtained in step (3) respectively with OD600 It is seeded to for 0.1 inoculum concentration in the nitrogenous water distribution of Bisphenol F, and accesses the gaseous mixture (volume ratio 3/7) being made up of oxygen and helium It is aerated, reaction system is subjected to encapsulation process after 10min to be aerated, the static gas wave refrigerator 2d in 35 DEG C of incubators.Produce Gas with gas chromatography (GC) determine, obtain N2And N2O concentration；Determine liquid portion total nitrogen, ammonia nitrogen, nitrate nitrogen and Asia The concentration of nitrate nitrogen, pellet fraction is handled with freeze-drying, the concentration of organic nitrogen in somatic cells is determined, by nitrogen Quantitative to calculate the metabolic characteristic for determining bacterial strain to ammonia nitrogen, more than 47% initial ammonia nitrogen can be converted into by obtained bacterial strain HAW6 N2, it is same with normal strains function phase before degeneration.

Claims (1)

1. a kind of rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading, it is characterised in that step It is as follows：
(1) domestication culture is carried out to the strain for needing rejuvenation：
(1.1) fluid nutrient medium I is prepared, the concentration proportioning of various materials is：(NH4)2SO40.1-0.5g/L, sodium citrate 1- 3g/L, sodium acetate 1-3g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O 0.2-0.8g/L、CaCl2 0.02-0.1g/L, pH are adjusted to 6.8-7.2；
(1.2) the nitrogenous water distribution of bisphenols is prepared, the concentration proportioning of various materials is：(NH4)2SO40.01-0.02g/L, bisphenols Material 0.2-0.5g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O 0.2-0.8g/L、CaCl2 0.02-0.1g/L, pH are adjusted to 6.8-7.2；
The nitrogenous water distribution of the fluid nutrient medium I and bisphenols of preparation is subjected to sterilization treatment；
(1.3) three class I liquid I culture mediums are aseptically prepared：
The first kind is the fluid nutrient medium I of 100% volume；
Equations of The Second Kind includes multiple fluid nutrient mediums, each by the nitrogenous water distribution of fluid nutrient medium I and bisphenols of different volumes ratio It is made into, and the volume ratio shared by fluid nutrient medium I is prepared by the order successively decreased, fluid nutrient medium I volume ratio scope For 90%~10%；Volume ratio shared by the nitrogenous water distribution of bisphenols is prepared by incremental order, the body of the nitrogenous water distribution of bisphenols Product proportion is 10%~90%；
3rd class is the nitrogenous water distribution of bisphenols of 100% volume；
The three class I liquid I culture mediums prepared are subjected to sterilization treatment；
(1.4) the heterotrophic nitrification aerobic denitrifying strain for needing rejuvenation is carried out into progressively domestication to cultivate, first by first kind liquid Body culture medium, domestication culture 1-2d；Change again in Equations of The Second Kind fluid nutrient medium, the second class I liquid I is accounted for from the nitrogenous water distribution of bisphenols The 10% of the volume of culture medium starts, and gradually increases the volume ratio of the nitrogenous water distribution of bisphenols, until the nitrogenous water distribution of bisphenols is accounted for The 90% of the volume of Equations of The Second Kind fluid nutrient medium, often increases once, domestication culture 1-2d；Finally change the 3rd class I liquid I culture Base, domestication culture 1-2d；
Tame the specific steps of culture：Aseptically, it is with phosphate buffer that the heterotrophic nitrification for needing rejuvenation is good first Oxygen denitrification strain is cleaned and centrifuged 2-3 times, removes supernatant；It is subsequently added into phosphate buffer and carries out concussion resuspension, obtains Uniform bacterium solution, pipettes bacterium solution and is seeded in the fluid nutrient medium of step (1.3) preparation so that the concentration of initial strain is OD600= 0.1-0.15, finally by fluid nutrient medium be placed in it is sterile, 30-35 DEG C, domestication culture is carried out under conditions of 100-150rpm；Its In, the concentration proportioning of each material is in phosphate buffer：K2HPO4·2H2O 5-6g/L、KH2PO42-3g/L, by phosphate PH of buffer is adjusted to 6.8-7.2；Centrifugal condition is：8000-10000×g、5min、4℃；OD600To be surveyed under 600nm wavelength The heterotrophic nitrification aerobic denitrifying bacterium bacteria concentration obtained；
(2) bacterium after domestication culture is isolated and purified：
Solid medium is prepared, the concentration proportioning of various materials is：(NH4)2SO40.1-0.5g/L, sodium citrate 1-3g/L, second Sour sodium 1-3g/L, K2HPO4·2H2O 5-6g/L、KH2PO4 2-3g/L、MgSO4·7H2O 0.2-0.8g/L、CaCl2 0.02- 0.1g/L, agar 20g/L, pH are adjusted to 6.8-7.2；
The heterotrophic nitrification aerobic denitrifying bacterium tamed after culture is seeded to sterilizing solid medium with plate streak, by flat board It is inverted in 30-35 DEG C of constant incubator and cultivates 2-3d to obtain single bacterium colony；
(3) primary dcreening operation is carried out to efficient bacterium：
A. the single bacterium colony in picking flat board is seeded in first kind fluid nutrient medium respectively, and the first class I liquid I after inoculation is trained Foster base is placed in 30-35 DEG C, isothermal vibration culture 1-2d under the conditions of 100-150rpm, and ammonia nitrogen and nitrogen removal rate is measured by sampling；
B. ammonia nitrogen and nitrogen removal rate level to measure reaches the bacterial strain of the normal removal level more than 80% of strain, with OD600For 0.1-0.15 inoculum concentration is seeded in the 3rd class I liquid I culture medium respectively, and the 3rd class I liquid I culture medium after inoculation is placed in 30-35 DEG C, isothermal vibration culture 2-3d under the conditions of 100-150rpm, are measured by sampling bis-phenol pollutant, ammonia nitrogen and total nitrogen and remove Rate；
Repeat step B, until bis-phenol pollutant, ammonia nitrogen and the total nitrogen removal of measure reach the preceding normal removal level of degeneration and protect It is fixed to keep steady；
(4) nitrogen balance analysis is carried out to efficient bacterium：By the bacterial strain obtained in step (3) with OD600For 0.1-0.15 inoculum concentration It is seeded in the nitrogenous water distribution of bisphenols, and accesses oxygen and helium volume ratio 3：The gaseous mixture of 7 compositions is aerated, and waits to be aerated Reaction system is subjected to encapsulation process after 10-20min, the static gas wave refrigerator 2-3d in 30-35 DEG C of incubator；The gas of generation is used Gas chromatography is determined, and obtains N2And N2O concentration；The concentration of liquid portion total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen is determined, Pellet fraction is handled with freeze-drying, the concentration of organic nitrogen in somatic cells is determined, is determined by the quantitative calculating to nitrogen Bacterial strain obtains the heterotrophic nitrification aerobic denitrifying bacterial strain of efficient degradation bisphenols nitric wastewater to the metabolic characteristic of ammonia nitrogen.
CN201710251089.2A 2017-04-19 2017-04-19 A kind of rejuvenation method of the heterotrophic nitrification aerobic denitrifying bacterium for bisphenols nitric wastewater of degrading Withdrawn CN107058207A (en)

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