CN105039212A - Rhodococcus strain C3, microbial agent containing rhodococcus strain C3, and applications of rhodococcus strain C3 and microbial agent - Google Patents
Rhodococcus strain C3, microbial agent containing rhodococcus strain C3, and applications of rhodococcus strain C3 and microbial agent Download PDFInfo
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Abstract
The invention discloses a rhodococcus strain C3. A preparation method of the rhodococcus strain C3 comprises the following steps of sucking an activated sludge sample from a wastewater biological treatment system of a coking plant to a fresh enrichment culture medium of phenol for shaking culture, wherein the inoculum concentration is 1%; after an enrichment bottle becomes muddy, taking a certain amount of the culture solution, inoculating a phenol liquid culture medium with increased concentration with the culture solution for culturing; in the same way, gradually increasing the concentration of phenol to 1000mg/L; inoculating an inorganic salt culture medium with the concentration of phenol being 1000mg/L with the strain enriched by the enrichment culture medium for domestication; and separating and screening a strain taking phenol as the unique carbon source and energy source from an isolation medium plate by adopting a dilution spread plate method and a streak plate A single colony of the strain is separated from an LB solid medium in a streaking manner, wherein the single colony is yellowish and semitransparent, has a neat edge and wet texture, and is easy to prick up; phenol, pyridine and o-cresol are taken as carbon sources, and the strain can degrade 1000mg/L of phenol in the inorganic salt liquid culture medium. The invention further provides a microbial agent taking rhodococcus strain C3 as the active component, and a preparation method of the microbial agent.
Description
Technical field
The invention belongs to environmental pollutant biological reinforcing technology field, be specifically related to a kind of rhodococcus and application rhodococcus strain C3 in the treatment of waste water thereof, be related specifically to the biological treatment of phenols organic pollutant.
Background technology
Phenol belongs to highly toxic substance, has strong corrosive nature to skin, mucous membrane, and can suppress nervus centralis or infringement Liver and kidney function, high-concentration phenol can make protein coagulating and cause tissue injury, necrosis.In water body, the phenol of 5 ~ 25mg/L can constitute a threat to the existence of fish.Because phenol has carcinogenic, teratogenesis, mutagenic genotoxic potential, each state all in succession proposes and phenol is classified as priority pollutants in water.Mainly come from oil refining, coking of coal factory, producer gas plant containing aldehydes matter, if this type of waste water is unprocessed and discharge in a large number, serious environmental pollution will be caused, serious harm HUMAN HEALTH and ecotope.Therefore, how effectively Phenol-Containing Wastewater Treatment is the problem that environmental pollution prevention and control field urgently solves.
The height of current investigation and application comprises distillation, liquid-liquid extraction, absorption, pervaporation, advanced oxidation and biological treatment etc. containing the wastewater processing technology of aldehydes matter.Biological treatment is comparatively economy, actual effect, pollution-free transfer, wield typical process technology easy and simple to handle, is also the main stream approach of current wastewater treatment.But due to the existence of high density aldehydes matter, cause the biologic treating techniques such as traditional activated sludge process, there is sludge acclimatization time long, the problem such as treatment effect is unstable, impact resistance is poor.And in sewage treatment facility, add efficient degrading bacteria, under the prerequisite of low cost, greatly can improve degradation efficiency.
In recent years, study the extraordinary microorganism in physical environment with competitive edge and become the new focus of water treatment.Domestic and international investigator is studied some efficient degrading bacterias, and such as, Rhodopseudomonas has the ability of metabolism multiple compounds, has quite high degrading activity to the such as compound such as arene, arene derivatives; Corynebacterium is the main bacteria seed of cracking heterogeneous ring compound and hydrocarbon chain; The oxidable hydrocarbon compound of genus arthrobacter, steroidal compounds etc.; These high-efficiency strains have broad application prospects in organic wastewater with difficult degradation thereby (TNT waste water, paper waste, waste water from dyestuff and coking chemical waste water etc.) process.
Embedded immobilization microbial Technological expression is the feature that microbe density is high, have anti-shock loading and wide adaptability.Its Growth and distribution can not change because administering the existence of substrate, and the embedded immobilization materials and methods adopted has again stronger specific aim.Therefore, microorganism embedded immobilization technology is a kind of important means microorganism being obtained more extensively, more effectively applies, and it will have broad application prospects in field of wastewater treatment.
Summary of the invention
An object of the present invention is the bacterial strain providing a highly effective degrading phenol, and the improvement for phenolic wastewater is provided fundamental basis and technical support.
Two of object of the present invention is to provide a kind of microbiobacterial agent utilizing above-mentioned rhodococcus to produce.
One strain rhodococcus strain C3, it is characterized in that, described bacterial strain is prepared by following step and obtains:
The activated sludge sample of a certain amount of Coking Plant Wastewater biological treatment system is drawn to shaking culture in the fresh enrichment medium of phenol by 1% inoculum size; After enrichment bottle muddiness, get a certain amount of nutrient solution and be transferred to increase in certain density liquid phenol substratum and cultivate; The like, improve phenol concentration step by step to 1000mg/L; By through phenol concentration be 1000mg/L enrichment medium enrichment after thalline switching phenol concentration be tame in 1000mg/L minimal medium; Adopt the method for dilution spread and line, on isolation medium flat board, separation screening take phenol as the bacterial strain of sole carbon source and the energy;
Described liquid enrichment medium component is peptone, extractum carnis, sodium-chlor, deionized water and phenol, wherein peptone: extractum carnis: sodium-chlor: the mass ratio of deionized water is 10:3:5:1000, and phenol concentration is added as required; Described inorganic salt liquid substratum take phenol as sole carbon source, and component and mass ratio are 0.5KH
2pO
4: 0.5K
2hPO
4: NH
4nO
3, 0.2MgSO
4: 0.2CaCl
2: 0.01FeSO
47H
2o:0.01MnSO
4h
2o, also comprises phenol, and phenol content is measured as required and added, adding distil water constant volume;
The bacterial strain prepared is accredited as Rhod Rhodococcussp. through 16SrDNA, and gramstaining is positive, and urase, catalase are positive, oxydase, VP reaction and clark and Lubsreaction feminine gender; On LB solid medium, line is separated single bacterium colony, yellowish translucent, neat in edge, and quality is moistening, easily provokes; Described rhodococcus strain C3 can with phenol, pyridine, ortho-cresol for carbon source, its 1000mg/L phenol of can degrading in inorganic salt liquid substratum.
Present invention also offers a kind of microbiobacterial agent utilizing above-mentioned rhodococcus strain C3 to produce, its active ingredient is above-mentioned rhodococcus strain C3.
Present invention also offers a kind of preparation method of the microbiobacterial agent utilizing above-mentioned rhodococcus strain C3 to produce, comprise the steps:
(1) described rhodococcus strain C33 is inoculated into enrichment medium 30 DEG C, under 200r/min condition, cultivates 24h-48h, to obtain the logarithmic phase cell of thalline after 7000rpm centrifugation 3-5min, and wash 2 ~ 3 times with phosphate buffered saline buffer;
(2) by centrifugal go out bacterium liquid add charcoal absorption, wherein gac add-on is 3 ~ 10wt%;
(3) PVA is added to the water, dissolve completely under 90 DEG C of water bath condition, the weight/volume of PVA and water is 1:8 ~ 10, add sodium alginate, silicon-dioxide and calcium carbonate after cooling and mix, PVA: sodium alginate: silicon-dioxide: the weight ratio of calcium carbonate is 10:3 ~ 6:35:0.2 ~ 0.5, obtains PVA gel-sodium alginate gel;
(4) after obtained described PVA-sodium alginate gel and the bacterium liquid adding gac being mixed, obtain thalline-PVA-sodium alginate suspension, what thalline-PVA-sodium alginate suspension low speed is instilled continuously stirring contains 3 ~ 5%CaCl
2boric acid solution in, described boric acid solution pH, in neutral, take out, forms immobilization particle after the crosslinked 24 ~ 90h of 4 ~ 6 DEG C of immobilizations;
(5) by immobilization particle in solidifying agent K
2hPO
4after solidification 2-8h, with deionized water wash 2 ~ 3 times, obtain immobilized microorganism preparation.
The present invention still further provides the application of this rhodococcus strain C3 in the biological treatment of coking chemical waste water or refinery water or tar production phenolic organic pollutants from wastewater.
The present invention still further provides the application of microbiobacterial agent of the present invention in Phenol-Containing Wastewater Treatment, comprising following operation: be that the concentration of 3%-10% adds in phenolic wastewater by weight with volume ratio by described microbiobacterial agent.。
The present invention compared with prior art, has the following advantages:
1) efficient degrading bacterial strain provided by the present invention comes from the active sludge of Coking Plant Wastewater biological treatment system, and through acclimating and screening, and obtained by plate streaking separation and purification, the toxic organic compound degradation efficiencies such as phenol are high.
2) to prepare the method for immobilized microorganism preparation easy in the present invention, and obtained immobilized microbial inoculum effective degradation bacteria concentration is high, thalline not easily runs off, shock resistance is strong.
3) compared with the biological reinforced treatment process directly adding thalline, there is the advantages such as specificity is strong, processing efficiency is high, stability is strong, bacterial classification is high-purity efficiently, sludge quantity is few, good effect of separating solid from liquid, operational management is simple, economy is better.
Accompanying drawing explanation
Fig. 1 is the effect of microbial inoculum to the degraded of coking phenol in wastewater;
Fig. 2 is the effect of microbial inoculum to the degraded of phenol in coking processing waste water;
Fig. 3 is the effect of microbial inoculum to the degraded of phenol in refinery water.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Embodiment 1: the acquisition of rhodococcus strain C3 bacterial strain and qualification
One, the acquisition of bacterial classification
Bacterial strain comes from the active sludge of Coking Plant Wastewater biological treatment system.Drawing 2ml mud sample by 1% inoculum size is in the fresh enrichment medium of 200mg/L to 200ml phenol concentration, shaking table shake shaking culture (30 DEG C, 200r/min).After enrichment bottle muddiness, getting 2ml nutrient solution, to be transferred to phenol concentration be cultivate in the liquid nutrient medium of 400mg/L.The like, improve phenol concentration step by step to 1000mg/L.By through phenol concentration be 1000mg/L enrichment medium enrichment after thalline switching phenol concentration be tame in 1000mg/L minimal medium.Adopt the method for dilution spread and line, on isolation medium flat board, separation screening take phenol as the bacterial strain of sole carbon source and the energy.
Beef-protein medium, specifically fills a prescription: peptone 10g, extractum carnis 3g, sodium-chlor 5g, deionized water 1000ml, phenol (amount adds as required).Inorganic salt liquid substratum take phenol as sole carbon source, specifically fills a prescription: KH
2pO
40.5g, K
2hPO
40.5g, NH
4nO
31.0g, MgSO
40.2g, CaCl
20.2g, FeSO
47H
2o0.01g, MnSO
4h
2o0.01g, phenol (amount adds as required), adding distil water is settled to 1L, and solid medium adds 18 ~ 20g agar in this substratum.
Two, the qualification of bacterial strain
(1) morphological specificity: thalline is rod-short
(2) physiological and biochemical property: G+ bacteria (Gram-positive); Urase, catalase are positive, oxydase, VP reaction and clark and Lubsreaction feminine gender; At LB cultured on solid medium after 3 days, bacterium colony size about 2 ~ 3mm, faint yellow, bacterium colony is protruding, and edge is irregular, bacterium colony surface wettability, translucent.
(3) 16SrDNA Sequence Identification
Bacterial strain 16SrDNA sequence alignment is identified: collected by centrifugation thalline, extracts STb gene with test kit.Use 16SrDNA forward primer F1:5 '-AGAGTTTGATCCTGGCTCAG-3 ', reverse primer R1:5 '-AAGGAGGTGATCCAGCCGCA-3 ' carries out DNA cloning (PCR).PCR reaction conditions: 95 DEG C of denaturation 5min, 95 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 90s, and 30 circulations, 72 DEG C finally extend 10min.The row that check order use NCBIBlast compare of analysis, result shows, with its sequence similarity reach 99% be Rhodococcussp., its 16SrDNA sequence is shown in sequence table.In conjunction with the physio-biochemical characteristics of bacterial strain, belonged to Rhod (Rhodococcussp.), be numbered C3.
Embodiment 2: rhodococcus strain RhodococcusC3 is to the degraded of pyridine, ortho-cresol in minimal medium
The mono-bacterium colony of picking rhodococcus strain C3 is in 3mlLB liquid nutrient medium, and 30 DEG C, 200r/min shaking culture 24 hours, obtain fresh bacterium liquid.Pyridine, ortho-cresol are added respectively in minimal medium, the final concentration of pyridine, ortho-cresol is respectively 100mg/L.Degradation results is in table 1.
| Substrate | Pyridine | Ortho-cresol |
| Degradation rate % | 100 | 100 |
Embodiment 3: rhodococcus strain C3 is to the degraded of coking phenol in wastewater, pyridine and ortho-cresol
Measure 100ml coking chemical waste water, the concentration of phenol is 350 ± 5mg/L; Interpolation pyridine, ortho-cresol make its concentration be respectively 100 ± 5mg/L, the fresh bacterium liquid of access 2ml, and mixing, is placed in 30 DEG C, and the shaking table of 200r/min is cultivated, the degradation rate of (120h) sampling and measuring compound after 5 days.The results are shown in Table 2.
| Substrate | Phenol | Pyridine | Ortho-cresol |
| Degradation rate % | 100 | >90 | >90 |
embodiment 4: prepared by immobilized microorganism microbial inoculum
Rhodococcus strain C3 is inoculated into enrichment medium 30 DEG C by 1, cultivates 24h under 200r/min condition, to obtain the logarithmic phase cell of thalline after 7000rpm centrifugation 5min, and washs 3 times with phosphate buffered saline buffer;
2. by centrifugal go out bacterium liquid add gac, absorption.Gac add-on is 6wt%.
3. PVA is added to the water, dissolve completely under 90 DEG C of water bath condition, the weight/volume of PVA and water is 1:10, add sodium alginate, silicon-dioxide and calcium carbonate after cooling and mix, PVA: sodium alginate: silicon-dioxide: the weight ratio of calcium carbonate is 10:5:3:0.2, obtains PVA gel-sodium alginate gel;
4. after above-mentioned obtained PVA-sodium alginate gel and the bacterium liquid adding gac being mixed, obtain thalline-PVA-sodium alginate suspension, what thalline-PVA-sodium alginate suspension low speed is instilled continuously stirring contains 5%CaCl
2boric acid solution in, described boric acid solution pH, in neutral, takes out after 4 DEG C of immobilizations are cross-linked 48h, with deionized water wash 3 times, obtains immobilization particle.
5. immobilization particle is solidified after 8h in solidifying agent K2HPO4, with deionized water wash 3 times, obtain immobilized microorganism preparation.
Embodiment 5: microbial inoculum is to the degraded of coking phenol in wastewater
Microbial inoculum is added in coking chemical waste water with the ratio of 5% (W/V), 4 groups of the effects microbial inoculums is set to the removal effect of coking phenol in wastewater:
A () adds 45mL coking chemical waste water, 50mL tap water, 5g microbial inoculum in 250mL Erlenmeyer flask;
B () adds 45mL coking chemical waste water, 50mL tap water, 5ml bacterium liquid in 250mL Erlenmeyer flask;
C () adds 45mL coking chemical waste water, 45mL tap water, 5g microbial inoculum and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
D () adds 45mL coking chemical waste water, 45mL tap water, 5g bacterium liquid and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
Be placed in 30 DEG C to cultivate with the shaking table of 200r/min, separated in time sampling detects phenol concentration in substratum, the results are shown in Figure 1.Result shows: although bacterial strain C3 can phenol effectively in treatment of Coking Wastewater, required time is longer, and after adding active sludge, the degradation rate of phenol and the equal decrease to some degree of degradation rate; Comparatively speaking, the microbial inoculum phenol degrading rate prepared by bacterial strain C3 is high, and can with other microorganism syntrophism in active sludge, and all reach more than 99% to the degradation rate of the phenol in coking chemical waste water in 24h, Screening of high efficient paracetamolum is remarkable.
Embodiment 7: rhodococcus strain C3 is to the degraded of phenol in coking processing waste water
Microbial inoculum is added in coking processing waste water with the ratio of 5% (W/V), 4 groups of the effects microbial inoculums is set to the removal effect of coking phenol in wastewater:
A () adds 45mL coking processing waste water, 50mL tap water, 5g microbial inoculum in 250mL Erlenmeyer flask;
B () adds 45mL coking processing waste water, 50mL tap water, 5ml bacterium liquid in 250mL Erlenmeyer flask;
C () adds 45mL coking processing waste water, 45mL tap water, 5g microbial inoculum and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
D () adds 45mL coking processing waste water, 45mL tap water, 5g bacterium liquid and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
Be placed in 30 DEG C to cultivate with the shaking table of 200r/min, separated in time sampling detects phenol concentration in substratum, the results are shown in Figure 2.Result shows: although bacterial strain C3 can phenol effectively in treatment of Coking Wastewater, required time is longer, and after adding active sludge, the degradation rate of phenol and the equal decrease to some degree of degradation rate; Comparatively speaking, the microbial inoculum phenol degrading rate prepared by bacterial strain C3 is high, and can with other microorganism syntrophism in active sludge, and reach more than 99% to the degradation rate of the phenol in coking chemical waste water in 24h, Screening of high efficient paracetamolum is remarkable.
Embodiment 8: rhodococcus strain C3 is to the degraded of phenol in refinery water
Microbial inoculum is added in refinery water with the ratio of 5% (W/V), 4 groups of the effects microbial inoculums is set to the removal effect of coking phenol in wastewater:
A () adds 45mL refinery water, 50mL tap water, 5g microbial inoculum in 250mL Erlenmeyer flask;
B () adds 45mL refinery water, 50mL tap water, 5ml bacterium liquid in 250mL Erlenmeyer flask;
C () adds 45mL refinery water, 45mL tap water, 5g microbial inoculum and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
D () adds 45mL refinery water, 45mL tap water, 5g bacterium liquid and the unpasteurized active sludge of 5mL in 250mL Erlenmeyer flask;
Be placed in 30 DEG C to cultivate with the shaking table of 200r/min, separated in time sampling detects phenol concentration in substratum, the results are shown in Figure 3.Result shows: although bacterial strain C3 can phenol effectively in treatment of Coking Wastewater, required time is longer, and after adding active sludge, the degradation rate of phenol and the equal decrease to some degree of degradation rate; Comparatively speaking, the microbial inoculum phenol degrading rate prepared by bacterial strain C3 is high, and can with other microorganism syntrophism in active sludge, and all reach more than 99% to the degradation rate of the phenol in coking chemical waste water in 24h, Screening of high efficient paracetamolum is remarkable.
Claims (5)
1. a strain rhodococcus strain C3, it is characterized in that, described bacterial strain is prepared by following step and obtains:
The activated sludge sample of a certain amount of Coking Plant Wastewater biological treatment system is drawn to shaking culture in the fresh enrichment medium of phenol by 1% inoculum size; After enrichment bottle muddiness, get a certain amount of nutrient solution and be transferred to increase in certain density liquid phenol substratum and cultivate; The like, improve phenol concentration step by step to 1000mg/L; By through phenol concentration be 1000mg/L enrichment medium enrichment after thalline switching phenol concentration be tame in 1000mg/L minimal medium; Adopt the method for dilution spread and line, on isolation medium flat board, separation screening take phenol as the bacterial strain of sole carbon source and the energy;
Described liquid enrichment medium component is peptone, extractum carnis, sodium-chlor, deionized water and phenol, wherein peptone: extractum carnis: sodium-chlor: the mass ratio of deionized water is 10:3:5:1000, and phenol concentration is added as required; Described inorganic salt liquid substratum take phenol as sole carbon source, and component and mass ratio are 0.5KH
2pO
4: 0.5K
2hPO
4: NH
4nO
3, 0.2MgSO
4: 0.2CaCl
2: 0.01FeSO
47H
2o:0.01MnSO
4h
2o, also comprises phenol, and phenol content is measured as required and added, adding distil water constant volume;
The bacterial strain prepared is accredited as Rhod Rhodococcussp. through 16SrDNA, and gramstaining is positive, and urase, catalase are positive, oxydase, VP reaction and clark and Lubsreaction feminine gender; On LB solid medium, line is separated single bacterium colony, yellowish translucent, neat in edge, and quality is moistening, easily provokes; Described rhodococcus strain C3 can with phenol, pyridine, ortho-cresol for carbon source, its 1000mg/L phenol of can degrading in inorganic salt liquid substratum.
2. the microbiobacterial agent of rhodococcus strain C3 production according to claim 1, is characterized in that its active ingredient is rhodococcus strain C3 according to claim 1.
3. a preparation method for the microbiobacterial agent described in claims 2, is characterized in that, comprises the steps:
(1) described rhodococcus strain C33 is inoculated into enrichment medium 30 DEG C, under 200r/min condition, cultivates 24h-48h, to obtain the logarithmic phase cell of thalline after 7000rpm centrifugation 3-5min, and wash 2 ~ 3 times with phosphate buffered saline buffer;
(2) by centrifugal go out bacterium liquid add charcoal absorption, wherein gac add-on is 3 ~ 10wt%;
(3) PVA is added to the water, dissolve completely under 90 DEG C of water bath condition, the weight/volume of PVA and water is 1:8 ~ 10, add sodium alginate, silicon-dioxide and calcium carbonate after cooling and mix, PVA: sodium alginate: silicon-dioxide: the weight ratio of calcium carbonate is 10:3 ~ 6:35:0.2 ~ 0.5, obtains PVA gel-sodium alginate gel;
(4) after obtained described PVA-sodium alginate gel and the bacterium liquid adding gac being mixed, obtain thalline-PVA-sodium alginate suspension, what thalline-PVA-sodium alginate suspension low speed is instilled continuously stirring contains 3 ~ 5%CaCl
2boric acid solution in, described boric acid solution pH, in neutral, take out, forms immobilization particle after the crosslinked 24 ~ 90h of 4 ~ 6 DEG C of immobilizations;
(5) by immobilization particle in solidifying agent K
2hPO
4after solidification 2-8h, with deionized water wash 2 ~ 3 times, obtain immobilized microorganism preparation.
4. the application of the rhodococcus strain C3 described in claims 1 in the biological treatment of coking chemical waste water or refinery water or tar production phenolic organic pollutants from wastewater.
5. the application of microbiobacterial agent according to claim 2 in Phenol-Containing Wastewater Treatment, is characterized in that, comprises following operation: be that the concentration of 3%-10% adds in phenolic wastewater by weight with volume ratio by described microbiobacterial agent.
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| CN108130288A (en) * | 2017-12-15 | 2018-06-08 | 浙江工业大学 | The application of Rhodococcus ruber and its degradable organic pollutant |
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