CN104152377A - Heavy-metal-tolerant aerobic denitrification strain and application thereof - Google Patents
Heavy-metal-tolerant aerobic denitrification strain and application thereof Download PDFInfo
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Abstract
The invention relates to a heavy-metal-tolerant aerobic denitrification strain and application thereof and belongs to the technical field of environment friendliness. The strain is Bacillus pumilus SNR-3 and preserved in China General Microbiological Culture Collection Center (referred to as CGMCC) and the preservation number is CGMCC NO. 7126. The strain disclosed by the invention can tolerate heavy metals such as Cd<2+> and Cu<2+> and can be subjected to aerobic denitrification in the presence of the heavy metals to effectively remove nitric nitrogen and nitrite nitrogen out of a water body and adsorb the corresponding heavy metals. The strain has the advantages of extensive culture conditions, simple and convenient operation and low treatment cost and has important significance for efficient purification of nitrogen-containing wastewater polluted by the heavy metals.
Description
Technical field
What the present invention relates to is a kind of biological treating method of environmental protection technical field, specifically a kind of aerobic denitrifying bacteria and application thereof that tolerates heavy metal.
Background technology
Underground water, as the important component part of water resources, plays a part very important in the evolution of human society.Over nearly 20 years, due to the unreasonable disposal of domestic waste and Industrial " three Waste " etc., a large amount of uses of agriculture production Pesticides, chemical fertilizer, national groundwater pollution situation increases the weight of increasingly.According to the sampling and testing data presentation of Chinese Geological office in 2010, the main Plain of east China groundwater quality totally causes anxiety, underground water three polluted by nitrogen are general, be facial contamination feature, its exceeding standard rate reaches 7.7～15.4%, more than 10 province of North of Huai River approximately has 3,000 ten thousand people to drink high nitric acid salt solution, and Haihe basin contaminated underground water stock number accounts for 62% of underground water total resources, and rural area approximately has 3.6 hundred million people not drink standard compliant tap water.Heavy metal detects and is generally point pollution feature, is distributed in surrounding city and industrial and mining enterprises dirty irrigated area around more.Safe drinking water and people's health have been arrived in the common combined pollution serious threat existing of this multi-pollutant.Therefore the underground water of repairing and administer three nitrogen, heavy-metal composite pollution is extremely urgent.
Most of based technique for in-situ remediation about groundwater pollution are all for certain first kinds of pollution matter now.For the improvement of nitrate pollution, biological denitrificaion is the most cost-effective improvement technology at present.Aerobic denitrifying bacteria is the microorganism that a class can be brought into play denitrification under aerobic conditions, thereby also can synchronize and carry out with nitrification, and avoids
to the inhibition of nitration reaction, accelerate nitration reaction process.Process at present in the method for heavy metal wastewater thereby biosorption process and be one of the most promising method.So-called biosorption process is exactly to utilize the chemical structure of some organism itself and composition characteristic to adsorb metal ion soluble in water, then is separated and is removed the method for metal ion in the aqueous solution by solid-liquid two.What in the report about biological adsorption, mention in recent years is all the adsorption of non-active cells, but it is practical that the biological adsorption of dead cell is difficult to, and for the metal of some kind, dead cell is difficult to active adsorption, and live body bacterium can be realized specific adsorption, so people start again to pay close attention to the utilization of living microorganism in the Heavy Metals Bio-adsorption.Metal ion enters viable cell, generally will pass through the outer combination of born of the same parents and be transported to two stages in born of the same parents.The former is a Fast Process, does not require the expenditure of energy, and is called passive adsorption; The latter carries out comparatively slowly, depends on the regulation and control of energy and metabolic system, is called active absorption.Although investigators have obtained larger progress at biological denitrificaion and biological adsorption field respectively, also lack the bioremediation technology research of relative synchronous reparation groundwater and heavy metal contamination.
Through the retrieval of prior art is found, Chinese patent literature CN103937701A open (bulletin) day 2014.07.23, a kind of bacillus pumilus shou002 and application thereof are disclosed, bacillus pumilus (Bacillus pumilus) shou002 is wherein preserved in Chinese Typical Representative culture collection center on November 3rd, 2013, deposit number is CCTCC NO:M2013536, this bacillus pumilus shou002 has good restraining effect to aquatic pathogenic bacterium, and no pathogenicity, safe and reliable, the effectively organism in degrading cultivation water, the pollutents such as ammonia nitrogen and nitrous acid nitrogen, Optimal culture environment, promote water surrounding benign ecological cycles, can be applied in aquaculture as fodder additives and water ecological setting modifying agent.But this technology does not relate to the degraded of water body nitric nitrogen, and bacterial strain itself do not have the ability of Adsorption of Heavy Metals, cannot be applied to the nitrogenous effluent of heavy metal contamination.
Li Han discloses two strain aerobic denitrifying bacterias in " Nitrogen Removal Mechanism of aerobic denitrifying bacteria and the research of flocculating properties thereof " (Institutes Of Technology Of Nanjing, environmental engineering, 2013, Master's thesis), respectively called after ADB4A and ADB7.Nitric nitrogen influent concentration be set be 268.8mgL ?1, at 160r/min, under the envrionment conditions of 30 DEG C, cultivate after 2 days, strains A DB4A and ADB7 have reached respectively 83% and 63% to the degradation rate of nitric nitrogen.Through 16SrDNA sequencing, think that strains A DB4A and ADB7 are respectively bacillus pumilus and Bacillus licheniformis.In order to understand better and to utilize aerobic denitrifying bacteria, the document is set about from factors such as carbon source, carbon-nitrogen ratio, temperature, rotating speed, pH, inquire into the optimal environmental condition of aerobic denitrifying bacteria growth and the impact of the aerobic denitrification of environment on bacterial strain by single factor method, all obtained optimum parameter.But this technology, water body nitric nitrogen being carried out to denitrification while processing, can cause the accumulation of nitrite nitrogen and ammonia nitrogen in water body, causes secondary pollution.
Summary of the invention
The present invention is directed to prior art above shortcomings, propose a kind of aerobic denitrifying bacteria and application thereof that tolerates heavy metal, this bacterial strain can tolerate Cd
2+, Cu
2+in heavy metal, and carry out aerobic denitrification under the condition that can exist at above-mentioned heavy metal, effectively remove nitric nitrogen and nitrite nitrogen in water body, and adsorb corresponding heavy metal.This strain culturing condition is extensive, and easy to operate simple, processing cost is low, and the high-efficient purification of the nitrogenous effluent to heavy metal contamination is significant.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of bacillus pumilus (Bacillus pumilus) SNR ?3, this bacterial strain is preserved in Chinese common micro-organisms culture presevation administrative center (being called for short CGMCC) at present, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101.Deposit number is CGMCC NO.7126, and preservation date is on January 11st, 2013.
Described bacillus pumilus SNR ?3 there is following character:
1, colonial morphology feature: vegetative cell is the bacillus of 0.4～0.8 × 1.5～2.7 μ m sizes on peptone nutrient agar, cultivates for 37 DEG C and forms gemma in 2～3 days, and gemma is Long Circle or cylindric.In above-mentioned substratum, 30 DEG C of cultivation 8h thalline can raised growth.Bacterium colony is white in color, and there is fold at edge, opaque, does not glisten.
2, physiology and biochemistry characteristic: culture temperature: 20～37 DEG C, optimum temperuture is 30 DEG C; In the scope of pH5.8～7.5, grow; Gramstaining tests positive; Methyl red experiment tests positive; Nitrate reduction tests positive; Indoles production testing is positive; H2S production testing is negative; Urase production testing is negative.
Described bacillus pumilus SNR ?3 record through 16S rDNA sequence, the most of sequence 1414bp of its 16S rDNA, as shown in SEQ ID No.1, gene order in this sequence and GenBank is carried out to homology comparison, find that itself and bacillus pumilus (Bacillus pumilus) homology reach 99.9%.
The present invention relates to a kind of bacillus pumilus (Bacillus pumilus) SNR ?3 application, use it for the nitric nitrogen and the nitrite nitrogen that remove in polluted-water, be specially: by bacillus pumilus SNR ?after 3 enlarged culturing, be inoculated in and contain Cd
2+and/or Cu
2+nitrogenous effluent in, thalline removes nitric nitrogen and the nitrite nitrogen in water body by aerobic denitrification in process of growth, utilizes biological adsorption effect to remove the Cd in water body simultaneously
2+, Cu
2+heavy metal, and the heavy metal being adsorbed is all positioned at born of the same parents, thus reach the object of removal heavy metal.
Described enlarged culturing refers to: by SNR ?3 inoculation in contain carbon source, nitrogenous source, inorganic salt and water without bacteria fermentation culture medium in, be more than or equal to 5 at C/N ratio, initial pH is 5.8～7.5, culture temperature is to cultivate 6～24 hours under the environment of 25～37 DEG C.
Described comprises following component without bacteria fermentation culture medium: carbon source 2～40g/L, nitrogenous source 0.1～20g/L, inorganic salt 0.01～20g/L, all the other are water, pH6.0～7.5, wherein: described carbon source is any one or a few the combination in glucose, sucrose, maltose, lactose, wood sugar, fructose, citric acid, glycerine and molasses; Nitrogenous source is extractum carnis, peptone, yeast extract paste, starch, corn steep liquor, soybean cake powder, cottonseed meal, urea, (NH
4)
2sO
4, KNO
3and NH4NO
3in any one or a few combination; Inorganic salt are one or more in vitriol, phosphoric acid salt, dihydrogen phosphate and hydrochloride.
Described inoculation refers to: the ratio according to 0.5～10% is put in nitrogenous effluent.
In described nitrogenous effluent, the starting point concentration of nitric nitrogen is that 10～2000mg/L, nitrite nitrogen starting point concentration are 1～350mg/L.
Cd in described nitrogenous effluent
2+starting point concentration be 0.01～10mg/L, Cu
2+starting point concentration be 0.01～30mg/L.
Technique effect
Compared with prior art, technique effect of the present invention comprises:
1) bacillus pumilus (Bacillus pumilus) that the present invention screens acquisition can utilize several kinds of carbon source and nitrogenous source to carry out growth and breeding, and culture condition is very extensive, easy to operate simple.
2) bacterial strain provided by the invention can carry out denitrification completely under aerobic condition, is nitrogen by nitric nitrogen direct-reduction, realizes thorough denitrogenation and can not cause secondary pollution to environment.
3) bacterial strain provided by the invention has Cd
2+, Cu
2+heavy metal tolerance, can pass through the biological adsorption effect heavy-metal ion removal of thalline self, and can synchronize and carry out with aerobic denitrification process, be applicable to process the nitrogenous effluent of nitrogenous effluent or heavy metal contamination, there is excellent environmental benefit and social benefit.
Brief description of the drawings
Fig. 1 be bacillus pumilus (Bacillus pumilus) SNR ?3 colonial morphology photo.
Fig. 2 be bacillus pumilus (Bacillus pumilus) SNR ?3 colony microscope photo (1000 ×).
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Bacillus pumilus (Bacillus pumilus) SNR ?3 separation and purifying
Enrichment medium 1: Seignette salt 20g/L, trisodium citrate 10g/L, sodium acetate 10g/L, KNO
32g/L, K
2hPO
40.5g/L, MgSO
47H
2o0.2g/L, trace element solution 0.2%, pH7.5.Trace element solution: EDTA-2Na57.1mg/L, ZnSO
47H
2o3.9mg/L, CaC1
22H
2o7mg/L, MnCl
24H
2o5.1mg/L, FeSO
47H
2o5.0mg/L, CuSO
45H
2o1.6mg/L, CoC1
21.6mg/L.
Enrichment medium 2: add 100mg/L CuSO45H2O on the basis of enrichment medium 1,50mg/L (CH
3cOO)
2cd2H
2o.
BTB substratum: KNO
31.0g/L, C
6h
5na
3o
22H
2o1.0g/L, KH
2pO
41.0/Lg, FeS0
47H
200.05g/L, CaCl
20.2g/L, MgSO
47H
2o1.0g, 1% BTB1mL, agar 20.0g/L, pH6.8.
Denitrification substratum: Seignette salt 20g/L, KNO
32.5g/L, K
2hPO
40.5g/L, MgSO
47H
2o0.2g/L, agar 20.0g/L, pH7.5.
From the coastal beach of Chongming Island gather activated sludge sample, the activated sludge 10g that gets respectively different sampling points adds in enrichment medium 1,30 DEG C, 150r/min are cultivated 3d, access in new enrichment medium 1 with 10% inoculum size, continue to cultivate according to the method described above, so repeatedly tame 4 times, with enrichment aerobic denitrifying bacteria.
Above-mentioned enrichment bacterium liquid is accessed in enrichment medium 2 again, carry out acclimating cultivation with identical method, repeat after 4 times, draw lmL enrichment culture liquid to 9mL stroke-physiological saline solution, obtain 10
-1extent of dilution bacteria suspension, is diluted to 10 by 10 times of dilution methods successively
-8.Draw respectively each extent of dilution suspension 0.lmL to BTB solid medium and be evenly coated with, be inverted for 30 DEG C and cultivate 2～3d, picking has single bacterium colony of blue haloing, and on denitrification solid medium, continuous passage cultivated for 6 generations, until obtain the stable pure culture of colonial morphology.
Embodiment 2
Bacillus pumilus (Bacillus pumilus) SNR ?3 denitrification effects to nitric wastewater
Seed culture medium: glucose 5g/L, extractum carnis 3g/L, peptone 5g/L, NaCl0.5g/L, utilizes NaOH solution to adjust pH6.5.
Fermention medium: glucose 20g/L, yeast extract paste 2g/L, peptone 5g/L, K
2hPO
43H
2o2.5g/L, MgSO
47H
2o0.1g/L, utilizes NaOH solution to adjust pH7.0.
By bacillus pumilus B.pumilus SNR ?3 (CGMCC NO.7126) under seed culture medium, 30 DEG C, the shaking table condition of 200r/min, cultivate 10h, seed liquor is inoculated in by the grain weight of 2% (v/v) in the 5L fermentor tank of the fermention medium being preinstalled with after 3.5L sterilizing and cultivates, culture condition: 30 DEG C, 300r/min, air flow 1.5L/min, fermenting process is without regulating pH.Fermentation 12h, cell concentration reaches 300 × 10
8cfu/mL.
Above-mentioned bacterium liquid is accessed respectively in the sewage that contains nitric nitrogen starting point concentration 300mg/L, nitrite nitrogen starting point concentration 100mg/L, wherein by the inoculum size of 4% (v/v): C/N ratio is 15.Under 25 DEG C, 300r/min, air flow 1.2L/min condition, cultivate 5d, get nutrient solution centrifugal 10min under 8000rpm condition, get supernatant and detect nitric nitrogen and nitrite nitrogen content.
The denitrification effect of table 1 bacterial strain to nitric wastewater
Embodiment 3
Bacillus pumilus (Bacillus pumilus) SNR ?3 to Cu
2+the repairing effect of the nitric wastewater polluting
Seed culture medium: glucose 5g/L, extractum carnis 3g/L, peptone 5g/L, MgSO
47H
2o0.3g/L, utilizes NaOH solution to adjust pH6.5.
Fermention medium: glucose 15g/L, sucrose 10g/L, peptone 5g/L, extractum carnis 3g/L, K
2hPO
43H
2o2.5g/L, MgSO
47H
2o0.1g/L, utilizes NaOH solution to adjust pH7.2.
By bacillus pumilus B.pumilus SNR ?3 (CGMCC NO.7126) under seed culture medium, 30 DEG C, the shaking table condition of 200r/min, cultivate 10h, seed liquor is inoculated in by the grain weight of 2% (v/v) in the 5L fermentor tank of the fermention medium being preinstalled with after 3.5L sterilizing and cultivates, culture condition: 35 DEG C, 300r/min, air flow 2.0L/min, fermenting process is without regulating pH.Fermentation 8h, cell concentration reaches 300 × 10
8cfu/mL.
Above-mentioned bacterium liquid is contained to nitric nitrogen starting point concentration 300mg/L, nitrite nitrogen starting point concentration 50mg/L, Cu by the inoculum size access of 6% (v/v)
2+in the sewage of starting point concentration 5mg/L, wherein C/N ratio is 20,25 DEG C, cultivate 5d when 300r/min, air flow 1.5L/min, get nutrient solution centrifugal 10min under 8000rpm condition, get supernatant and detect nitric nitrogen, nitrite nitrogen and Cu
2+content.
Table 2 bacterial strain is to Cu
2+the repairing effect of the nitric wastewater polluting
Embodiment 4
Bacillus pumilus (Bacillus pumilus) SNR ?3 to Cd
2+the repairing effect of the nitric wastewater polluting
Seed culture medium: sucrose 5g/L, yeast powder 3g/L, peptone 5g/L, MgSO47H2O0.3g/L, utilizes NaOH solution to adjust pH6.8.
Fermention medium: glucose 20g/L, glycerine 5g/L, peptone 5g/L, (NH
4)
2sO
42g/L, K
2hPO
43H
2o2.5g/L, MgSO
47H
2o0.1g/L, utilizes NaOH solution to adjust pH7.2.
By bacillus pumilus B.pumilus SNR ?3 (CGMCC NO.7126) under seed culture medium, 30 DEG C, the shaking table condition of 200r/min, cultivate 10h, seed liquor is inoculated in by the grain weight of 4% (v/v) in the 5L fermentor tank of the fermention medium being preinstalled with after 3.5L sterilizing and cultivates, culture condition: 35 DEG C, 400r/min, air flow 1.5L/min, fermenting process is without regulating pH.Fermentation 8h, cell concentration reaches 300 × 10
8cfu/mL.
Above-mentioned bacterium liquid is contained to nitric nitrogen starting point concentration 500mg/L, nitrite nitrogen starting point concentration 30mg/L, Cd by the inoculum size access of 6% (v/v)
2+in the sewage of starting point concentration 5mg/L, wherein C/N ratio is 20,25 DEG C, cultivate 5d when 300r/min, air flow 1.5L/min, get nutrient solution centrifugal 10min under 8000rpm condition, get supernatant and detect nitric nitrogen, nitrite nitrogen and Cu
2+content.
Table 3 bacterial strain is to Cd
2+the repairing effect of the nitric wastewater polluting
Embodiment 5
Bacillus pumilus (Bacillus pumilus) SNR ?3 to Cu
2+, Cd
2+the repairing effect of the nitric wastewater polluting
Seed culture medium: glucose 5g/L, extractum carnis 3g/L, peptone 5g/L, MgSO
47H
2o0.3g/L, utilizes NaOH solution to adjust pH6.5.
Fermention medium: glucose 20g/L, sucrose 10g/L, peptone 5g/L, (NH
4)
2sO
43g/L, K
2hPO
43H
2o2.5g/L, MgSO
47H
2o0.1g/L, utilizes NaOH solution to adjust pH7.2.
By bacillus pumilus B.pumilus SNR ?3 (CGMCC NO.7126) under seed culture medium, 30 DEG C, the shaking table condition of 200r/min, cultivate 10h, seed liquor is inoculated in by the grain weight of 4% (v/v) in the 5L fermentor tank of the fermention medium being preinstalled with after 3.5L sterilizing and cultivates, culture condition: 30 DEG C, 400r/min, air flow 1.8L/min, fermenting process is without regulating pH.Fermentation 8h, cell concentration reaches 300 × 10
8cfu/mL.
Above-mentioned bacterium liquid is contained to nitric nitrogen starting point concentration 600mg/L, nitrite nitrogen starting point concentration 50mg/L, Cu by the inoculum size access of 10% (v/v)
2+starting point concentration 10mg/L, Cd
2+in the sewage of starting point concentration 2mg/L, wherein C/N ratio is 25,25 DEG C, cultivate 5d when 300r/min, air flow 1.5L/min, get nutrient solution centrifugal 10min under 8000rpm condition,
Get supernatant and detect nitric nitrogen, nitrite nitrogen, Cu
2+, Cd
2+content.
Table 4 bacterial strain is to Cu
2+, Cd
2+the repairing effect of the nitric wastewater polluting
Claims (9)
1. a bacillus pumilus SNR ?3, it is characterized in that, this bacillus pumilus (Bacillus pumilus) is preserved in Chinese common micro-organisms culture presevation administrative center, and deposit number is CGMCC NO.7126, and preservation date is on January 11st, 2013.
Bacillus pumilus SNR according to claim 1 ?3, it is characterized in that, described bacillus pumilus SNR ?3 record sequence as shown in SEQ ID No.1 through 16S rDNA sequence.
3. an application of bacillus pumilus SNR ?3 according to claim 1 and 2, is characterized in that, uses it for the nitric nitrogen and the nitrite nitrogen that remove in polluted-water, is specially: by after bacillus pumilus SNR ?3 enlarged culturing, be inoculated in and contain Cd
2+and/or Cu
2+nitrogenous effluent in, thalline removes nitric nitrogen and the nitrite nitrogen in water body by aerobic denitrification in process of growth, utilizes biological adsorption effect to remove the Cd in water body simultaneously
2+, Cu
2+heavy metal, and the heavy metal being adsorbed is all positioned at born of the same parents, thus reach the object of removal heavy metal.
4. application according to claim 3, it is characterized in that, described enlarged culturing refers to: by SNR ?3 inoculation in contain carbon source, nitrogenous source, inorganic salt and water without bacteria fermentation culture medium in, be more than or equal to 5 at C/N ratio, initial pH is 5.8～7.5, culture temperature is to cultivate 6～24 hours under the environment of 25～37 DEG C.
5. application according to claim 3, is characterized in that, described comprises following component without bacteria fermentation culture medium: carbon source 2～40g/L, and nitrogenous source 0.1～20g/L, inorganic salt 0.01～20g/L, all the other are water, pH6.0～7.5.
6. application according to claim 5, is characterized in that, described carbon source is any one or a few the combination in glucose, sucrose, maltose, lactose, wood sugar, fructose, citric acid, glycerine and molasses; Nitrogenous source is extractum carnis, peptone, yeast extract paste, starch, corn steep liquor, soybean cake powder, cottonseed meal, urea, (NH
4)
2sO
4, KNO
3and NH
4nO
3in any one or a few combination; Inorganic salt are one or more in vitriol, phosphoric acid salt, dihydrogen phosphate and hydrochloride.
7. application according to claim 3, is characterized in that, described inoculation refers to: the ratio according to 0.5～10% is put in nitrogenous effluent.
8. application according to claim 3, is characterized in that, in described nitrogenous effluent, the starting point concentration of nitric nitrogen is that 10～2000mg/L, nitrite nitrogen starting point concentration are 1～350mg/L.
9. application according to claim 3, is characterized in that, Cd in described nitrogenous effluent
2+starting point concentration be 0.01～10mg/L, Cu
2+starting point concentration be 0.01～30mg/L.
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| CN108977400A (en) * | 2018-09-18 | 2018-12-11 | 宁夏医科大学 | A kind of bacillus pumilus culture medium |
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