CN103146604B - Comamonas testosteroni LH-N5 and heterotrophic nitrification-aerobic denitrification microbial inoculum, and preparation method and application thereof - Google Patents
Comamonas testosteroni LH-N5 and heterotrophic nitrification-aerobic denitrification microbial inoculum, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Comamonas testosteroni LH-N5 CGMCC No.6975. The invention also discloses a heterotrophic nitrification-aerobic denitrification microbial inoculum which comprises the Comamonas testosteroni LH-N5, or one or both of Achromobacter xylosoxidans subtype xylosoxidans LH-N25 and Paracoccus aminovorans LH-N40; and the strains are combined in any proportion. The invention also discloses a preparation method of the microbial inoculum. The microbial inoculum disclosed by the invention can solve the problem of difficulty in removing total nitrogen in the traditional reactor, can effectively remove ammonia nitrogen and total nitrogen in the water body in one reactor, has tolerance or degradation capability for phenols, amines, heterocycles, cyanides, polycyclic aromatic hydrocarbons and other toxic substances in wastewater, is especially applicable to nitrogenous chemical wastewater, has the advantages of simple treatment process, stable effect and environmental toxicant impact resistance, and has very wide application prospects in various actual nitrogenous chemical wastewater treatment processes.
Description
Technical field
The invention belongs to environmental microorganism field, be specifically related to microbial strains, and the microbiobacterial agent of the heterotrophic nitrification aerobic denitrifying denitrogenation being formed by this microbial strains, and the preparation method of this microbial inoculum and purposes.
Background technology
Traditional wastewater biological denitrificaion technology is mainly to lean on Autotrophic nitrification bacterium and heterotrophic denitrifying Bacteria to make ammonia nitrogen change nitrogen into by nitrification and denitrification coupling to remove.And the rate of propagation of autotrophic bacteria self is slow, cannot to compete with heterotrophic organism, be difficult to obtain higher biomass, nitrification efficiency in the Sludge System of mixed culture low, cause that autotrophic microorganism denitrification system impact resistance is weak, nitrification is incomplete; Denitrifying bacteria is heterotrophic bacterium, in servicely often needs to add carbon source, has increased running cost.
Deficiency in view of traditional nitrobacteria and denitrifying bacterium, it is long start time that the discovery of allotrophic nitrobacteria, aerobic denitrifying bacteria has solved traditional biological denitrogenation processing, nitrated link conditional request is harsh, and nitrification and denitrification such as can not synchronously carry out at the shortcoming, has good development prospect.The allotrophic nitrobacteria of having reported much has again the function of aerobic denitrification simultaneously, and this introduces new concept for sewage water denitrification, and synchronous nitration and denitrification is achieved.
At present, the waste water of high ammonia nitrogen industry generally contains a lot containing hazardous and noxious substances, as coking chemical waste water etc.For this situation, obtain the efficient heterotrophic nitrification aerobic denitrifying function stem that tenable environment is poisoned, removed hard-degraded substance, structure is combined into microbiobacterial agent, be added in Waste Water Treatment or for the biological restoration of specific polluted-water, become the dominant population in reaction system and bring into play denitrogenation and remove COD effect, can strengthen the degradation capability to difficult degradation pollutent, improve denitrification rates, and improve the removal usefulness of original biological treatment system to target contaminant.
Patent document CN101875909.B provides a kind of efficient heterotrophic nitrification aerobic denitrifying bacteria and cultural method and purposes, this bacterium is Rhod Rhodococcus sp.DN2.3, preservation registration number is CCTCC M209300, ammonia nitrogen in can effective elimination water body, nitrous acid nitrogen, nitrate nitrogen and composition thereof also can be removed the COD in organic waste water simultaneously
cr, but microbial inoculum production method is too simple, and bacterial classification is single, and environment tolerance is poor, is not suitable for extensive utilization.
Patent document CN102277314.B provides the efficient allotrophic nitrobacteria screening method of kind, the bacterial strain of screening and the preparation method of microbial inoculum thereof, this bacterial strain is bacillus cereus (Bacillus cereus), deposit number is: CGMCCNO.4918, can utilize nitrite to carry out growth and breeding as unique nitrogenous source.And prepare purification of water quality microbial preparation microbial inoculum with this bacterial strain, and can be used for the water such as sewage disposal, aquaculture, aquarium or mobile water system and administer engineering, have no it and process for industrial sewage.
Patent document CN101899401.A provides a kind of ammonia-containing water microbiobacterial agent and preparation method thereof, this microbial inoculum comprises Nitromonas, Nitrosomas and denitrifying bacteria, by orientation, tame to regulate or proportioning, can effectively process low COD, high ammonia-nitrogen wastewater, but the bacterial strain comprising in microbial inoculum is too general, poor to hazardous and noxious substances tolerance.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and a kind of new Comamonas testosteroni LH-N5 is provided.
Another technical problem to be solved by this invention has been to provide the heterotrophic nitrification aerobic denitrifying microbiobacterial agent that contains above-mentioned Comamonas testosteroni LH-N5.
Another technical problem to be solved by this invention has been to provide preparation method and the purposes of above-mentioned microbial inoculum.
The present invention is a kind of Comamonas testosteroni (Comamonas testosteroni) LH-N5 CGMCC No. 6975.
Bacterial strain involved in the present invention is Comamonas testosteroni (Comamonas testosteroni) LH-N5 CGMCC No. 6975, Achromobacter xylosoxidans wood sugar oxidation subspecies (Achromobacter xylosoxidans subsp. xylosoxidans) LH-N25 CGMCC No. 6972 and bites secondary coccus (Paracoccus aminovorans) the LH-N40 CGMCC of ammonia No. 6971.Wherein: Comamonas testosteroni is ensconced the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms December 10 in 2012, and deposit number is CGMCC No. 6975; Depositary institution address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, phone: 010-64807355; Achromobacter xylosoxidans wood sugar oxidation subspecies LH-N25 is deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms December 10 in 2012, and deposit number is CGMCC No. 6972; Depositary institution address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, phone: 010-64807355; Bite the secondary coccus LH-N40 of ammonia and December 10 in 2012, be deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No. 6971; Depositary institution address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, phone: 010-64807355.
Below the separation and purification of bacterial strain of the present invention is described.
One, the separation and purification of Comamonas testosteroni LH-N25
1. soil sample domestication enrichment: get pedotheque, be seeded in nitrated liquid nutrient medium, progressively tame enrichment.(nitrated enrichment medium forms: NH
4sO
41g/L, NaNO
31g/L, NaCl 0.3g/L, FeSO
40.03g/L, K
2hPO
40.25g/L, MgSO
40.03g/L, Na
2cO
30.3g/L, CH
3cOONa 0.5g/L distilled water 1L, pH 8.0)
2. diluent is prepared: get acclimation sludge nutrient solution 1ml, add in the sterilizing test tubes that fills 9ml sterilized water, mix and obtain 10
-1the mud suspension of concentration.Standing 30s, draws 10 with 1ml aseptic straw
-1mud suspension 1ml put into the aseptic water pipe of 9ml, pressure-vaccum mixes, and is 10
-2diluent, successively from 10
-2serial dilution to 10
-5, obtain a series of 10 times of diluents.
3. culture dish is prepared: get sterilizing culture dish, at ware, cover filling sample number, substratum code name, inoculation extent of dilution, separated date etc.Pour in ware the nitrated substratum after sterilizing into approximately 12～15ml, make the flat board that congeals into.(nitrated solid medium forms with nitrated liquid nutrient medium, separately adds 2% agar).
4. primary dcreening operation and cultivation: primary dcreening operation inoculation adopts coating method, draws different multiples diluent 0.1ml with aseptic straw, drips on the flat board having made, and then with aseptic spatula, smoothens.Inoculate completely, ware is classified overlapping, be inverted in 30 ℃ of incubators and cultivate 2～4d.
5. sieve again: take out cultured flat board, select growth well, representational single bacterium colony, use transfering loop picking, being inoculated into 30 ℃ of concussions in the nitrated liquid nutrient medium of same sample ingredient cultivates, after growth, with the transfering loop picking one ring nutrient solution purifying of ruling on nitrated solid medium, in 30 ℃ of incubators, cultivate.After having bacterium colony to grow, the line of picking list bacterium colony, purifying, repeats 2～3 times, and screening obtains pure bacterial strain.
6. this pure bacterial strain is delivered to biotechnology (Shanghai) Co., Ltd. and carry out 16s rDNA order-checking, through being accredited as Comamonas testosteroni (Comamonas testosteroni).
Two, bite the separation and purification of the secondary coccus LH-N40 of ammonia
1. soil sample domestication enrichment: get pedotheque, be seeded in nitrated liquid nutrient medium, progressively tame enrichment.(nitrated enrichment medium forms: NH
4sO
41g/L, NaNO
31g/L, NaCl 0.3g/L, FeSO
40.03g/L, K
2hPO
40.25g/L, MgSO
40.03g/L, Na
2cO
30.3g/L, CH
3cOONa 0.5g/L distilled water 1L, pH 8.0)
2. diluent is prepared: get acclimation sludge nutrient solution 1ml, add in the sterilizing test tubes that fills 9ml sterilized water, mix and obtain 10
-1the mud suspension of concentration.Standing 30s, draws 10 with 1ml aseptic straw
-1mud suspension 1ml put into the aseptic water pipe of 9ml, pressure-vaccum mixes, and is 10
-2diluent, successively from 10
-2serial dilution to 10
-5, obtain a series of 10 times of diluents.
3. culture dish is prepared: get sterilizing culture dish, at ware, cover filling sample number, substratum code name, inoculation extent of dilution, separated date etc.Pour in ware the nitrated substratum after sterilizing into approximately 12～15ml, make the flat board that congeals into.(nitrated solid medium forms with nitrated liquid nutrient medium, separately adds 2% agar).
4. primary dcreening operation and cultivation: primary dcreening operation inoculation adopts coating method, draws different multiples diluent 0.1ml with aseptic straw, drips on the flat board having made, and then with aseptic spatula, smoothens.Inoculate completely, ware is classified overlapping, be inverted in 30 ℃ of incubators and cultivate 2～4d.
5. sieve again: take out cultured flat board, select growth well, representational single bacterium colony, use transfering loop picking, being inoculated into 30 ℃ of concussions in the nitrated liquid nutrient medium of same sample ingredient cultivates, after growth, with the transfering loop picking one ring nutrient solution purifying of ruling on nitrated solid medium, in 30 ℃ of incubators, cultivate.After having bacterium colony to grow, the line of picking list bacterium colony, purifying, repeats 2～3 times, and screening obtains pure bacterial strain.
6. this pure bacterial strain is delivered to biotechnology (Shanghai) Co., Ltd. and carry out 16s rDNA order-checking, through being accredited as, bite the secondary coccus (Paracoccus aminovorans) of ammonia.
Three, the separation and purification of Achromobacter xylosoxidans wood sugar oxidation subspecies LH-N25
1. soil sample domestication enrichment: get pedotheque, be seeded in denitrification liquid nutrient medium, progressively tame enrichment.(denitrification enrichment medium forms NaNO
31g/L, NaNO
20.5g/L, NaCl 0.3g/L, FeSO
40.03g/L, K
2hPO
40.25g/L, MgSO
40.03g/L, Na
2cO
30.3g/L, CH
3cOONa 0.5g/L distilled water 1L, pH 8.0)
2. diluent is prepared: get acclimation sludge nutrient solution 1ml, add in the sterilizing test tubes that fills 9ml sterilized water, mix and obtain 10
-1the mud suspension of concentration.Standing 30s, draws 10 with 1ml aseptic straw
-1mud suspension 1ml put into the aseptic water pipe of 9ml, pressure-vaccum mixes, and is 10
-2diluent, successively from 10
-2serial dilution to 10
-5, obtain a series of 10 times of diluents.
3. culture dish is prepared: get sterilizing culture dish, at ware, cover filling sample number, substratum code name, inoculation extent of dilution, separated date etc.Pour in ware the denitrification substratum after sterilizing into approximately 12～15ml, make the flat board that congeals into.(denitrification solid medium forms with denitrification liquid nutrient medium, separately adds 2% agar).
4. primary dcreening operation and cultivation: primary dcreening operation inoculation adopts coating method, draws different multiples diluent 0.1ml with aseptic straw, drips on the flat board having made, and then with aseptic spatula, smoothens.Inoculate completely, ware is classified overlapping, be inverted in 30 ℃ of incubators and cultivate 2～4d.
5. sieve again: take out cultured flat board, select growth well, representational single bacterium colony, use transfering loop picking, being inoculated into 30 ℃ of concussions in the denitrification liquid nutrient medium of same sample ingredient cultivates, after growth, with the transfering loop picking one ring nutrient solution purifying of ruling on denitrification solid medium, in 30 ℃ of incubators, cultivate.After having bacterium colony to grow, the line of picking list bacterium colony, purifying, repeats 2～3 times, and screening obtains pure bacterial strain.
6. this pure bacterial strain is delivered to biotechnology (Shanghai) Co., Ltd. and carry out 16s rDNA order-checking, through being accredited as Achromobacter xylosoxidans wood sugar oxidation subspecies (Achromobacter xylosoxidans subsp. xylosoxidans).
The invention also discloses a kind of heterotrophic nitrification aerobic denitrifying microbiobacterial agent, be characterized in: this microbiobacterial agent comprises described Comamonas testosteroni (Comamonas testosteroni) LH-N5; Or except comprising described Comamonas testosteroni (Comamonas testosteroni) LH-N5, also comprise Achromobacter xylosoxidans wood sugar oxidation subspecies (Achromobacter xylosoxidans subsp. xylosoxidans) LH-N25 CGMCC No. 6972 and bite one or both in secondary coccus (Paracoccus aminovorans) the LH-N40 CGMCC of ammonia No. 6971, between bacterial strain, according to ratio arbitrarily, combining.In microbial inoculum of the present invention, each bacterial strain is the bacterial strain that plays denitrogenation and remove COD effect in microbial inoculum, can also contain conventional microorganism aid, such as auxiliary agents such as stablizer and/or protective materials in microbial inoculum.
In microbiobacterial agent of the present invention, the content of every kind of bacterial strain need to be selected according to its handled waste water, and usually, with massfraction metering, the content of every kind of bacterial strain is all not less than 0.1%, is preferably not less than 1%, is most preferably not less than 5%.
The preparation method who the invention also discloses a kind of heterotrophic nitrification aerobic denitrifying microbiobacterial agent as described in above scheme, is characterized in, its step is as follows:
(1) bacterial strain described in microbiobacterial agent being inoculated into respectively to solid medium activates;
(2) after activation, be transferred to respectively respective liquid substratum and carry out shake-flask culture, centrifugal collection thalline, preparation high density seed liquor; Shake-flask culture condition is, pH:6.0～10.0, and temperature: 10 ℃～35 ℃, rotating speed: 100～170rpm, incubation time: 1～2 d; Living bacteria count amount>=5 * 10 in high density seed liquor
9cfu/mL;
(3) above-mentioned high density seed liquor is divided be clipped to and be inoculated into corresponding first class seed pot and carry out amplification culture 1～7d, culture condition is: 10 ℃～35 ℃ of temperature; PH 6.0～10.0; DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, stirring velocity 110～150rpm, the concentrated thalline of collecting, preparation primary seed solution; Again primary seed solution is transferred to respectively to corresponding secondary seed tank and carries out amplification culture 1～14d, the concentrated thalline of collecting, preparation secondary seed solution; Secondary seed solution is transferred to respectively to corresponding three grades of seeding tanks again and carries out amplification culture 1～21d, the concentrated thalline of collecting, obtains three grades of seed liquor; The culture condition of secondary seed solution and three grades of seed liquor is: pH 6.0～10.0,10～35 ℃ of temperature, DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, stirring velocity 110～150rpm; In three grades of seed liquor, add microorganism aid, according to the required ratio of each bacterial strain, mix and obtain microbiobacterial agent respectively; Described microorganism aid is stablizer and/or protective material;
Solid medium and liquid nutrient medium described in step (1) and (2) consist of: extractum carnis 3～5g/L, and peptone 6～12g/L, sodium-chlor 3～6g/L, distilled water 1L, adds 1.5～2.5% agar again in solid medium; The described primary seed solution substratum used of step (3) consists of: (NH
4)
2sO
40.5～5g/L, glucose 0.5～1g/L, MgSO
40.01～0.02g/L, K
2hPO
40.1～0.3g/L, FeSO
47H
2o 0.05～0.1 g/L, distilled water 1L; Secondary seed solution and three grades of seed liquor substratum used consist of: urea 0.5～5g/L, glucose 1～3g/L, K
2hPO
40.1～0.3g/L, MgSO
40.01～0.02g/L, FeSO
47H
2o 0.05～0.1 g/L, distilled water 1L.
In above-described preparation method's step (3): described stablizer and protective material can be conventional stablizer and the protective material using of normal bacteriostatic agent in prior art, and stablizer is preferably from a kind of of glycerine, ethanol or its mixture; Protective material is preferably from active carbon powder, diatomaceous a kind of or its mixture.Concentration method described in step (3) is preferably natural subsidence, centrifugal or filtration.
The microbiobacterial agent that microbiobacterial agent of the present invention or preparation method of the present invention make can be applied in processing nitrogenous wastewater from chemical industry.Microbiobacterial agent of the present invention can be prepared into liquid microbial inoculum, or is prepared into the microbial inoculum of dry powder.
Compared with prior art, beneficial effect of the present invention is as follows:
1. microbiobacterial agent of the present invention can not only solve the problem that in traditional reactor, total nitrogen difficulty removes, and can be in same reactor the ammonia nitrogen in effective elimination water body and total nitrogen; This microbiobacterial agent has tolerance or degradation capability to toxic substances such as the phenols in waste water, amine, heterocyclic, cyanogen class, cyanogen class, polycyclic aromatic hydrocarbonss; This microbiobacterial agent is conducive to the large-scale production application of the microbiobacterial agent of heterotrophic nitrification aerobic denitrifying denitrogenation; Can directly apply in the biological treatment system of nitrogenous organic chemical waste water; Can be used as biological reinforced dose and be added to Waste Water Treatment or biofilm aftertreatment waste water on various fillers; COD in can effective elimination waste water
cr, effect stability, resistance to environmental poisonous substance impacts; In processing, various nitrogenous organic chemical waste waters have boundless application prospect.
2. microbiobacterial agent preparation method of the present invention makes microbiobacterial agent viable count is high, with short production cycle, simple process.
Embodiment
Below in conjunction with specific examples, the present invention will be described, and following embodiment is only for the present invention is described, and be not used in restriction the present invention scope required for protection.
Embodiment 1, a kind of Comamonas testosteroni (Comamonas testosteroni) LH-N5 CGMCC No. 6975.
Embodiment 2, a kind of heterotrophic nitrification aerobic denitrifying microbiobacterial agent, and this microbiobacterial agent comprises the Comamonas testosteroni LH-N5 described in embodiment 1, and this microbial inoculum can be pure microbial inoculum, also contains conventional microorganism aid.Described microbiobacterial agent can be applied to process nitrogenous wastewater from chemical industry.
Embodiment 3, a kind of heterotrophic nitrification aerobic denitrifying microbiobacterial agent, this microbiobacterial agent comprises the Comamonas testosteroni LH-N5 described in embodiment 1, and Achromobacter xylosoxidans wood sugar oxidation subspecies LH-N25 CGMCC No. 6972 with bite a kind of in the secondary coccus LH-N40 of ammonia CGMCC No. 6971, between bacterial strain, according to ratio arbitrarily, combine.This microbial inoculum can be pure microbial inoculum, also contains conventional microorganism aid.Described microbiobacterial agent can be applied to process nitrogenous wastewater from chemical industry.
Embodiment 4, a kind of heterotrophic nitrification aerobic denitrifying microbiobacterial agent, this microbiobacterial agent comprises the Comamonas testosteroni LH-N5 described in embodiment 1, and Achromobacter xylosoxidans wood sugar oxidation subspecies LH-N25 CGMCC No. 6972 with bite the secondary coccus LH-N40 of ammonia CGMCC No. 6971, between bacterial strain, according to ratio arbitrarily, combine.This microbial inoculum can be pure microbial inoculum, also contains conventional microorganism aid.Described microbiobacterial agent can be applied to process nitrogenous wastewater from chemical industry.
Embodiment 5, and in described heterotrophic nitrification aerobic denitrifying microbiobacterial agent, with massfraction metering, the content of every kind of bacterial strain is all not less than 0.1% described in embodiment 2 or 3 or 4.
Embodiment 6, and in described heterotrophic nitrification aerobic denitrifying microbiobacterial agent, with massfraction metering, the content of every kind of bacterial strain is all not less than 5% described in embodiment 2 or 3 or 4.
Embodiment 7, and a kind of as the preparation method of the heterotrophic nitrification aerobic denitrifying microbiobacterial agent as described in any one in embodiment 2-6, its step is as follows:
(1) bacterial strain described in microbiobacterial agent being inoculated into respectively to solid medium activates;
(2) after activation, be transferred to respectively respective liquid substratum and carry out shake-flask culture, centrifugal collection thalline, preparation high density seed liquor; Shake-flask culture condition is, pH:6.0～10.0, and temperature: 10 ℃～35 ℃, rotating speed: 100～170rpm, incubation time: 1～2 d; Living bacteria count amount>=5 * 10 in high density seed liquor
9cfu/mL;
(3) above-mentioned high density seed liquor is divided be clipped to and be inoculated into corresponding first class seed pot and carry out amplification culture 1～7d, culture condition is: 10 ℃～35 ℃ of temperature; PH 6.0～10.0; DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, stirring velocity 110～150rpm, the concentrated thalline of collecting, preparation primary seed solution; Again primary seed solution is transferred to respectively to corresponding secondary seed tank and carries out amplification culture 1～14d, the concentrated thalline of collecting, preparation secondary seed solution; Secondary seed solution is transferred to respectively to corresponding three grades of seeding tanks again and carries out amplification culture 1～21d, the concentrated thalline of collecting, obtains three grades of seed liquor; The culture condition of secondary seed solution and three grades of seed liquor is: pH 6.0～10.0,10～35 ℃ of temperature, DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, stirring velocity 110～150rpm; In three grades of seed liquor, add microorganism aid, according to the required ratio of each bacterial strain, mix and obtain microbiobacterial agent respectively; Described microorganism aid is stablizer and/or protective material;
Solid medium and liquid nutrient medium described in step (1) and (2) consist of: extractum carnis 3～5g/L, and peptone 6～12g/L, sodium-chlor 3～6g/L, distilled water 1L, adds 1.5～2.5% agar again in solid medium; The described primary seed solution substratum used of step (3) consists of: (NH
4)
2sO
40.5～5g/L, glucose 0.5～1g/L, MgSO
40.01～0.02g/L, K
2hPO
40.1～0.3g/L, FeSO
47H
2o 0.05～0.1 g/L, distilled water 1L; Secondary seed solution and three grades of seed liquor substratum used consist of: urea 0.5～5g/L, glucose 1～3g/L, K
2hPO
40.1～0.3g/L, MgSO
40.01～0.02g/L, FeSO
47H
2o 0.05～0.1 g/L, distilled water 1L.
Embodiment 8, in the step (3) of the preparation side described in embodiment 7: described stablizer is selected from a kind of of glycerine, ethanol or its mixture; Described protective material is selected from active carbon powder, diatomaceous a kind of or its mixture.
Embodiment 9, and the concentration method described in the step (3) of the preparation side described in embodiment 7 or 8 is natural subsidence, centrifugal or filter.
Embodiment 10, the heterotrophic nitrification aerobic denitrifying performance test of microbiobacterial agent of the present invention.
One, preparation ammonia nitrogen mass concentration is the simulated wastewater of 240mg/L left and right, and interpolation methyl alcohol is carbon source, and COD mass concentration is 1050mg/L left and right.
Two, the pure bacterial strain fermentation liquor of preparing by the following method:
1. bacterial strain activation: 3 kinds of pure bacterial strains are received in solid medium, activated in 25 ℃ of constant incubators.
2. high density seed liquor preparation: after getting activation with transfering loop, bacterial strain is transferred in liquid nutrient medium, 10～35 ℃, 100～170rpm shaking flask concussion are cultivated 1～2d and are grown to logarithmic phase, in sterilizing centrifuge tube, the centrifugal supernatant liquor that goes is collected thalline, preparation high density seed liquor, living bacteria count amount>=5 * 10
9cfu/mL.Substratum consists of: extractum carnis 4g/L, and peptone 10g/L, sodium-chlor 5g/L, pH 7.5, and solid medium adds 2% agar.
3. primary seed solution preparation: above-mentioned high density seed liquor is divided to be clipped to be inoculated into 20L first class seed pot, 10 ℃～35 ℃ of temperature; PH 6.0～10.0; DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, amplification culture 3d under stirring velocity 110～150rpm condition, filtering and concentrating is collected thalline, preparation primary seed solution.Substratum consists of: (NH
4)
2sO
42g/L, glucose 1g/L, MgSO
40.01g/L, K
2hPO
40.2 g/L, FeSO
47H
2o 0.05 g/L.
4. secondary seed solution preparation: the primary seed solution of 3 strain bacterium is inoculated into 300L secondary seed tank amplification culture 10d, the concentrated thalline of collecting of natural subsidence, preparation secondary seed solution with 10% inoculum size respectively.Culture condition is pH 6.0～10.0,10～35 ℃ of temperature, DO 0.5～4.0mg/L, air flow 0.4～0.6m
3/ h, stirring velocity 110～150rpm.Substratum consists of: urea 4g/L, glucose 3g/L, K
2hPO
40.2g/L, MgSO
40.02g/L, FeSO
47H
2o 0.1 g/L.
5. three grades of seed liquor preparation: respectively the secondary seed solution of 3 strain bacterium is transferred to corresponding 5m with 10% inoculum size respectively
3three grades of seeding tank amplification culture 12d, the concentrated thalline of collecting of natural subsidence, obtains three grades of seed liquor.Culture condition and substratum form with secondary seed solution preparation condition.
Three. microbial inoculum preparation: by adding microorganism aid glycerine, mix and obtain microbiobacterial agent according to required ratio.
Wherein:
Microbial inoculum 1 is only containing Comamonas testosteroni LH-N5 bacterial classification in microbial inoculum;
It is formulated that microbial inoculum 2 is that Comamonas testosteroni LH-N5 and Achromobacter xylosoxidans wood sugar oxidation subspecies LH-N25 presses massfraction 1:1;
Microbial inoculum 3 is for Comamonas testosteroni LH-N5 is with to bite the secondary coccus LH-N40 of ammonia massfraction 1:1 formulated;
Microbial inoculum 4 is that Comamonas testosteroni LH-N5, Achromobacter xylosoxidans wood sugar are oxidized subspecies LH-N25 and bite the secondary coccus LH-N40 of ammonia massfraction 1:1:1 formulated.
Respectively microbial inoculum 1-4 is inoculated in 6L aeration reactor, inoculum size 10%, timing sampling is measured the variation of ammonia nitrogen, nitrate nitrogen, nitrous acid nitrogen concentration.The results are shown in Table 1, as can be seen from Table 1, through the cultivation of 12 hours, the ammonia nitrogen degradation rate in the system of microbial inoculum 1, microbial inoculum 2, microbial inoculum 3, microbial inoculum 4 reached respectively 91.06%, 92.97%, 93.04%, 94.17%, illustrated that four class microbial inoculums all have good removal effect to ammonia nitrogen.Simultaneously, at first 6 hours that cultivate, in microbial inoculum 1, microbial inoculum 2, microbial inoculum 3, microbial inoculum 4 nutrient solutions, all have micro-nitrate and nitrite to occur, nitrate and nitrite accumulation within 8 hours, do not detected again, in four class microbial inoculum systems, nitrogen removal rate all reaches more than 90.0% later.Illustrate that four class microbial inoculums have all realized heterotrophic nitrification aerobic denitrifying simultaneous denitrification.
The removal effect of table 1 denitrification microorganism microbial inoculum to ammonia nitrogen
Example 11, the application experiment of microbiobacterial agent in acrylic nitrile waste water.
One, acrylic nitrile waste water is taken from certain chemical plant, COD 2320～3540mg/L, and total nitrogen 295～480mg/L, ammonia nitrogen 210～385 mg/L, pH 8.5～10.0.
Two, preparation is containing the solid medium of acrylic nitrile waste water, Comamonas testosteroni LH-N5, Achromobacter xylosoxidans wood sugar are oxidized to subspecies LH-N25 and bite the secondary coccus LH-N40 of ammonia tri-strain bacterium bacterium liquid and be uniformly coated on acrylic nitrile waste water flat board, 25 ℃ of constant temperature culture 2d, investigate the growing state of three strain bacterium on acrylic nitrile waste water flat board.Result proves, three strain bacterium equal well-grown on acrylic nitrile waste water flat board, and otherness is less, and viable count is all 5 * 10
8～5 * 10
9within the scope of cfu/mL.
Three, with reference to above-mentioned experimental result, getting embodiment 10 makes microbial inoculum 1-4 and is inoculated in respectively in 6L sbr reactor device, do not inoculate other active sludge, passing into vinyl cyanide wastewater from chemical industry processes, flooding quantity 2～3.5L, 25～30 ℃ of temperature, pH 7.0～10.0, DO 2.0～4.0mg/L, without adding alkali lye.A cycle of operation comprises into water-aeration-precipitation-draining, and the whole service cycle is 12h, the 1～2h of intaking, and aeration 6～10h, precipitates 1～2h, draining 0.5h controls HRT(hydraulic detention time) 20.6～36h.Through 8 periodic duties, water outlet maintains COD 50～80mg/L, ammonia nitrogen 5～15mg/L, and total nitrogen 25～35mg/L, all reaches industry integrated wastewater discharge standard.
Embodiment 12, microbiobacterial agent and the comparative experiments of active sludge treatment waste water effect.
One, processing waste water is fumaric acid comprehensive wastewater, takes from certain chemical company, former water COD mass concentration: 20000～22000mg/L, ammonia nitrogen mass concentration: 1070～1190mg/L, total nitrogen concentration 1240～1380 mg/L, pH6.0～7.5, dilute before processing.
Two, test is carried out in 5 10L bio-aeration pools, first 5 aeration tanks add same concentrations denitrifying activated sludge, wherein the microbiobacterial agent 1-4 that embodiment 9 makes is added in 4 aeration tanks, and inoculum size is 20%, and same volume active sludge is added in another aeration tank.5 aeration tanks add the fumaric acid comprehensive wastewater of same volume, 25 ℃ of test temperatures, and pH 8.0, dissolved oxygen 2.0～3.0mg/L, treatment time 36h.The results are shown in Table 2, as can be seen from Table 2, add four class microbial inoculums the treatment effect of waste water is obviously better than to active sludge, wherein microbial inoculum 4 adds active sludge optimum, and the clearance specific activity mud method of ammonia nitrogen, total nitrogen and COD has been improved respectively to 38.97%, 44.48% and 32.16%.Four kinds of combined denitrification microbiobacterial agents not only have higher removal efficiency to ammonia nitrogen and total nitrogen in same reactor, and can tolerate higher COD, and the clearance of COD is all reached more than 90%.
Table 2 add microbial inoculum with not with microbial inoculum to the comparison of fumaric acid comprehensive wastewater result
Embodiment 13, the processing experiment of microbiobacterial agent to coking chemical waste water.
One, coking chemical waste water: take from certain coke-oven plant, this waste water complicated component, not only contain the inorganic pollutants such as ammonia, cyanogen, thiocyanate, also contain heterocycle and the polycyclc aromatic compounds such as phenol, naphthalene, pyridine, quinoline, carbazole, terphenyl, naphthalene, anthracene, poisonous hard-degraded substance content is high, and ammonia nitrogen concentration is high.Common materialization, biochemical process place except efficiency low, water outlet is not up to standard.First microbial inoculum Treatment of Wastewater in Coking passes through physico-chemical pretreatment, water inlet total nitrogen 350～450mg/L after processing, and ammonia nitrogen 250～330mg/L, pyridine 80～100mg/L, COD 1800～2200mg/L left and right, pH 9.0～10.0.
Two, first investigate the growing state of 3 strain bacterium on coking chemical waste water flat board, method is as described in embodiment 11.Experimental result is: LH-N5, LH-N25 growing state otherness are less, and viable count is 2 * 10
8～3 * 10
8cfu/mL, the viable count of LH-N40 on coking chemical waste water flat board is 5 * 10
8～2 * 10
9cfu/mL.
Three, with reference to above-mentioned experimental result, by method described in embodiment 10, prepare microbial inoculum, make respectively following microbial inoculum:
Microbial inoculum 2, with embodiment 10;
Microbial inoculum 5 is for Comamonas testosteroni LH-N5 is with to bite the secondary coccus LH-N40 of ammonia massfraction 2:1 formulated;
Microbial inoculum 6 is that Comamonas testosteroni LH-N5, Achromobacter xylosoxidans wood sugar are oxidized subspecies LH-N25 and bite the secondary coccus LH-N40 of ammonia massfraction 2:2:1 formulated.
Respectively microbial inoculum 2,5,6 is inoculated in 300L A/O reactor, dissolved oxygen 0～0.8mg/L, O pond dissolved oxygen 1～4mg/L are controlled in running-course control A pond, maintaining return sludge ratio is 3:1～4:1, temperature: 10 ℃～35 ℃, pH:6.0～10.0, each cycle 24h.
Microbial inoculum 2 operation 15d systems reach treatment capacity at full capacity, and water outlet total nitrogen is stabilized in 39～55mg/L, ammonia nitrogen≤15mg/L, pyridine 0.2～0.8mg/L, COD 80～100mg/L.
Microbial inoculum 5 operation 13d systems reach treatment capacity at full capacity, and water outlet total nitrogen is stabilized in 35～51mg/L, ammonia nitrogen≤15mg/L, and pyridine degradable is complete, COD 60～90mg/L.
Microbial inoculum 6 operation 11d systems reach treatment capacity at full capacity, and water outlet total nitrogen is stabilized in 33～45mg/L, ammonia nitrogen≤15mg/L, and pyridine degradable is complete, COD 50～80mg/L.
Three microbial inoculums all have good degradation effect to coking chemical waste water, and stable water outlet is up to standard, illustrate that this microbiobacterial agent is after adapting to the organic composition toxicity of coking chemical waste water complexity, can effectively utilize the organism in waste water that COD is reduced, and have good denitrification effect.
Claims (7)
- A Comamonas testosteroni ( comamonas testosteroni) LH-N5 CGMCC No. 6975.
- 2. a heterotrophic nitrification aerobic denitrifying microbiobacterial agent, is characterized in that: this microbiobacterial agent comprise Comamonas testosteroni claimed in claim 1 ( comamonas testosteroni) LH-N5; Or except comprise Comamonas testosteroni claimed in claim 1 ( comamonas testosteroni) outside LH-N5, also comprise Achromobacter xylosoxidans wood sugar oxidation subspecies (Achromobacter xylosoxidans subsp. xylosoxidans) LH-N25 CGMCC No. 6972 with bite the secondary coccus of ammonia ( paracoccus aminovorans) one or both in LH-N40 CGMCC No. 6971, between bacterial strain, according to ratio arbitrarily, combine.
- 3. heterotrophic nitrification aerobic denitrifying microbiobacterial agent according to claim 2, is characterized in that: in this microbiobacterial agent, with massfraction metering, the content of every kind of bacterial strain is all not less than 5%.
- 4. a preparation method for heterotrophic nitrification aerobic denitrifying microbiobacterial agent as claimed in claim 2 or claim 3, is characterized in that, its step is as follows:(1) bacterial strain described in microbiobacterial agent being inoculated into respectively to solid medium activates;(2) after activation, be transferred to respectively respective liquid substratum and carry out shake-flask culture, centrifugal collection thalline, preparation high density seed liquor; Shake-flask culture condition is, pH:6.0～10.0, and temperature: 10 ℃～35 ℃, rotating speed: 100～170rpm, incubation time: 1～2 d; Living bacteria count amount>=5 * 10 in high density seed liquor 9cfu/mL;(3) above-mentioned high density seed liquor is divided be clipped to and be inoculated into corresponding first class seed pot and carry out amplification culture 1～7d, culture condition is: 10 ℃～35 ℃ of temperature; PH 6.0～10.0; DO 0.5～4.0mg/L, air flow 0.4～0.6m 3/ h, stirring velocity 110～150rpm, the concentrated thalline of collecting, preparation primary seed solution; Again primary seed solution is transferred to respectively to corresponding secondary seed tank and carries out amplification culture 1～14d, the concentrated thalline of collecting, preparation secondary seed solution; Secondary seed solution is transferred to respectively to corresponding three grades of seeding tanks again and carries out amplification culture 1～21d, the concentrated thalline of collecting, obtains three grades of seed liquor; The culture condition of secondary seed solution and three grades of seed liquor is: pH 6.0～10.0,10～35 ℃ of temperature, DO 0.5～4.0mg/L, air flow 0.4～0.6m 3/ h, stirring velocity 110～150rpm; In three grades of seed liquor, add microorganism aid, according to the required ratio of each bacterial strain, mix and obtain microbiobacterial agent respectively; Described microorganism aid is stablizer and/or protective material;Solid medium and liquid nutrient medium described in step (1) and (2) consist of: extractum carnis 3～5g/L, and peptone 6～12g/L, sodium-chlor 3～6g/L, distilled water 1L, adds 1.5～2.5% agar again in solid medium; The described primary seed solution substratum used of step (3) consists of: (NH 4) 2sO 40.5～5g/L, glucose 0.5～1g/L, MgSO 40.01～0.02g/L, K 2hPO 40.1～0.3g/L, FeSO 47H 2o 0.05～0.1 g/L, distilled water 1L; Secondary seed solution and three grades of seed liquor substratum used consist of: urea 0.5～5g/L, glucose 1～3g/L, K 2hPO 40.1～0.3g/L, MgSO 40.01～0.02g/L, FeSO 47H 2o 0.05～0.1 g/L, distilled water 1L.
- 5. preparation method according to claim 4, is characterized in that, in step (3): described stablizer is selected from a kind of of glycerine, ethanol or its mixture; Described protective material is selected from active carbon powder, diatomaceous a kind of or its mixture.
- 6. preparation method according to claim 4, is characterized in that, the concentration method described in step (3) is natural subsidence, centrifugal or filtration.
- 7. the microbiobacterial agent that the preparation method described in the microbiobacterial agent described in claim 2 or 3 or claim 4 or 5 or 6 makes is purposes in processing nitrogenous wastewater from chemical industry.
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| CN106119159B (en) * | 2016-06-29 | 2019-07-23 | 西安交通大学 | The comamonas of one high-efficiency degradation pyridine carboxylic acid and its application |
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