CN104195066A - Azo dye orange II degrading bacterium and inocula produced from same - Google Patents

Abstract

The invention discloses an azo dye orange II degrading bacterium and an inocula produced from the same. The bacterial strain is bacterial strain L-11 of microbacterium oxydans, is preserved in China General Microbiological Culture Collection Center since 8th, May, 2014, the preservation address is No. 3, No. 1 yard, Beichen West Road, Chaoyang District, Beijing City, and the preservation number is CGMCC NO.9130. The main biological characteristics comprise that the bacterium colony is circle and is not transparent, the bacterium colony is white after being cultured for 1-3 days and is yellow after being continuously cultured, the liquid culture color is yellow, the optimum culture temperature is 28-30 DEG C, and the optimum pH is 7.2. The bacterium belongs to actinomyces. The degrading bacterium L-11 is capable of enabling the azo dye orange II residual quantity to be reduced by 80% or more. The azo dye orange II degrading bacterium inocula has the advantages of low production cost, usage convenience and good removing effect, and is applicable to pollution treatment of water and soil.

Description

The microbial inoculum of a kind of azoic dyestuff gold orange II degradation bacteria and production thereof

Technical field

The invention belongs to biological technical field, be specifically related to the microbial inoculum of a kind of azoic dyestuff gold orange II degradation bacteria and production thereof, is to utilize method of microorganism degradation of dye, is applicable to the control of environmental pollution.

Background technology

Microorganism is because kind is many, and metabolic type is various, has great advantage, especially microbiological treatment azoic dyestuff in dye wastewater treatment using than physics, chemical process.Under aerobic condition, thalline is metabolism azoic dyestuff well; Under anaerobic condition, a lot of microbe species electrophilic azo bond that can rupture, becomes 2 kinds of colourless aromatic amines by azo reductase by Degradation of Azo Dyes.Under these aromatic amine anaerobic conditions, can not further degrade, and poisonous to animal body, carcinogenic.But effective substrate that aromatic amine is aerobic biodegradation can further be degraded to the materials such as phenol under aerobic condition, if aerobic-anaerobic technique is used for wastewater treatment, aromatic amine can be by oxidative pathway open loop mechanism under aerobic condition by thorough mineralising.

The chromophore of azo molecules is azo bond, and auxochromes is amino, hydroxyl, methyl and sulfonic group etc., and after light is injected, generation selectivity absorbs and produces the shades of colour that vision is experienced.The initial decoloring reaction of azoic dyestuff biological decolouring process is the azo bond fracture [16] of carrying out under the katalysis of azo reductase.Decolorization can be carried out under aerobic condition, but anaerobic condition can significantly promote decolorization.Decoloring reaction can be represented by the formula:

To having, in the screening of microorganism of decolorizing azo dye waste water ability, oneself obtains a plurality of bacterial strains, Decolourization Bacteria majority is from sludge of sewage treatment plant or is subject to obtain dyestuff contaminated soil.As Xu Wendong etc., separation from the processing woollen mill waste water from dyestuff active sludge domestication obtains a strain degraded Black Liquor with Efficient Bacteria, through identifying that this bacterium is thermophilic Sphingobacterium.Luo Zhiteng, from long-term dyeing waste-water contaminated soil, screens 1 saccharomycete and can make only nitrogen source with direct orange 8.Liu Shenghao etc. adopt the method for enrichment culture, and from soil, separation obtains the Penicillium notatum P-93 that a strain azo dyes acid scarlet (GR) and erie black (ATT) have stronger decoloring ability.

Can the degrade Microbial resources of pyrazoles of occurring in nature are constantly found, and the intensive treatment that these Microbial resources are refractory organic industrial sewage is laid a good foundation.Azoic dyestuff gold orange II product water soluble, the pollution of azoic dyestuff gold orange II to environment, the comprehensive regulation of its environment of health of the mankind in serious harm, and the exploitation of the degradation bacteria of azoic dyestuff gold orange II in environment is there are no the technology of comparative maturity.

Summary of the invention

An object of the present invention is to provide a kind of azoic dyestuff gold orange II degradation bacteria.

Azoic dyestuff gold orange II degradation bacteria provided by the invention, its bacterial strain is the bacterial strain L-11 of oxidation microbacterium Microbacterium oxydans, on May 8th, 2014, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC NO.9130, Main Biological is that bacterium colony circle is opaque, cultivating 1～3 day bacterium colony is white, continuing to cultivate bacterium lives as yellow, liquid culture is yellow, cultivate 28～30 ℃ of optimum temperutures, pH7.2; These Pseudomonas actinomycetes.

Another object of the present invention provides the method for utilizing above-mentioned azoic dyestuff gold orange II degradation bacteria to produce microbial inoculum, and its technique is: inclined-plane kind-shake-flask seed liquid-seeding tank-product (packing formulation is liquid bacterial agent).

The concrete steps of the method are:

Step (1). the test tube kind of azoic dyestuff gold orange II degradation bacteria is inoculated in fermention medium, and shaking culture is to logarithmic phase;

Step (2). above-mentioned cultured bacterial classification is inoculated to the seeding tank into 500L by the inoculum size of 10 ﹪, be cultured to logarithmic phase, the culture medium prescription that seeding tank is used and mass content are: peptone 0.5 ﹪, yeast extract paste 0.25 ﹪, and with distilled water preparation, pH is 7.0;

Step (3). seed liquor is produced to tank by 10 ﹪ inoculum size accesses and cultivate, produce tank used medium identical with seed tank culture base;

Step (4). in the culturing process of seeding tank and production tank, the air flow of sterile air is 1:0.6～1.2, stirring velocity is 180～240r/min, culture temperature is 30 ℃, whole technical process incubation time is 48～60h, after fermentation ends thalline quantity reach 1,000,000,000/more than ml, the rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid agent, and this liquid agent is microbial inoculum.

Concrete beneficial effect of the present invention:

It is low, easy to use that azoic dyestuff gold orange II degradation bacterial agent of the present invention has production cost, and the advantage that removal effect is good is applicable to the Pollution abatement of water and soil.The present invention is for preserving the ecological environment, and to protect mankind is healthy, and the added value that improves agricultural-food has great importance.Degradation bacteria L-11 can make more than the residual quantity of azoic dyestuff gold orange II reduces by 80 ﹪.L-11 bacterial strain can be take azoic dyestuff gold orange II and grown as carbon source and the energy, and azoic dyestuff gold orange II degraded at short notice.

Accompanying drawing explanation

Fig. 1 is the impact of inoculum size on L-11 degradation efficiency;

Fig. 2 is the impact of initial pH on L-11 degradation efficiency;

Fig. 3 is the impact of temperature on L-11 degradation efficiency.

Embodiment

Below in conjunction with specific embodiment and accompanying drawing thereof, the present invention is further analyzed.

Azoic dyestuff gold orange II degradation bacteria, its bacterial strain is the bacterial strain L-11 of oxidation microbacterium Microbacterium oxydans, on May 8th, 2014, be preserved in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC NO.9130.

1. the separated and evaluation of bacterial strain

Get contaminated soil 10g, be placed in the sterilized water of 100ml, vibration 5min, obtains soil bacteria suspension; Get the soil bacteria suspension of 5ml, join the minimal medium [NH of 100ml ₄nO ₃: 407mg/L; KH ₂pO ₄: 98mg/L; K ₂hPO ₄: 33mg/L; NaCl:30mg/L; MgSO ₄: 200mg/L; PH7.0] in, add gold orange II as carbon source, be placed in 30 ℃, 150r/min shaking table is cultivated and is obtained pregnant solution, is coated with continuously, until obtain the degradation bacteria strains of gold orange II on the culture medium flat plate that adds gold orange II.This degradation bacteria called after L-11, is further purified and is accredited as oxidation microbacterium Microbacterium oxydans.L-11 bacterial strain Main Biological bacterium colony circle is opaque, and cultivating 1～3 day bacterium colony is white, continues to cultivate bacterium and lives as yellow, and liquid culture is yellow.Cultivate 28～30 ℃ of optimum temperutures, pH7.2.These Pseudomonas actinomycetes.The gold orange II of take grows as carbon source, and optimum growth temperature is 30 ℃.More than under the shake-flask culture condition of laboratory, the degradation rate of gold orange II reaches 75 ﹪.This bacterium can produce by the general fermentation equipment of fermentation industry.

2. laboratory biological degradation experiment

The impact of 2.1 inoculum sizes on L-11 degradation efficiency

Bacterial strain L-11 is at LB substratum (LB culture medium prescription g/L: yeast powder 5g/L, peptone 10g/L, NaCl10g/L, pH7.0) in be cultured to logarithmic phase, by the inoculum size of 0.5 ﹪, 1 ﹪, 2 ﹪, 3 ﹪, 4 ﹪, 5 ﹪, be inoculated into respectively nutrient solution [minimal medium [NH ₄nO ₃: 407mg/L; KH ₂pO ₄: 98mg/L; K ₂hPO ₄: 33mg/L; NaCl:30mg/L; MgSO ₄: 200mg/L; PH7.0], be placed in 30 ℃, the cultivation of 150r/min shaking table, after cultivation, 24h measures respectively nutrient solution in the OD at 600nm place value.As shown in Figure 1, inoculum size is larger for result, and the degradation efficiency of gold orange II is just higher; When being greater than 2 ﹪, inoculum size increasing inoculum size on gold orange II degraded impact not quite.Although the increment of thalline, along with inoculum size increases and improves, is considered thalli growth situation and economic reason, adopts the inoculum size of 1 ﹪ can reach requirement.

2.2pH the impact on L-11 degradation efficiency

PH value is another the important environmental factors that affects microorganism growth and existence, and microorganism growth has a suitable pH value scope, surpasses the pH value scope of suitable growth, the growth that most of microbe all can not fine people.

Bacterial strain L-11 is cultured to logarithmic phase in LB substratum (LB culture medium prescription g/L: yeast powder 5g/L, peptone 10g/L, NaCl10g/L, pH7.0); By the inoculum size of 1 ﹪, be inoculated into respectively the nutrient solution of seven kinds of different pH, wherein nutrient solution [minimal medium [NH ₄nO ₃: 407mg/L; KH ₂pO ₄: 98mg/L; K ₂hPO ₄: 33mg/L; NaCl:30mg/L; MgSO ₄: 200mg/L; PH7.0] pH value is adjusted to respectively 4.0,6.0,7.0,8.0,10.0; Be placed in 30 ℃, the cultivation of 150r/min shaking table, after cultivation, 8h, 16h, 24h and 32h measure respectively nutrient solution in the OD at 600nm place value.PH on the impact of bacterial strain L-11 growth as shown in Figure 2.Result shows: the appropriate pH value scope of L-11 strain growth is 7～10, and optimal ph is 7～8.

The impact of 2.3 temperature on L-11 degradation efficiency

Temperature is one of most important environmental factors affecting microorganism growth and existence.Bacterial strain L-11 is cultured to logarithmic phase in LB substratum (LB culture medium prescription g/L: yeast powder 5g/L, peptone 10g/L, NaCl10g/L, pH7.0), by the inoculum size of 1 ﹪, is inoculated into nutrient solution [minimal medium [NH ₄nO ₃: 407mg/L; KH ₂pO ₄: 98mg/L; K ₂hPO ₄: 33mg/L; NaCl:30mg/L; MgSO ₄: 200mg/L; PH7.0], be placed in respectively at 18 ℃, 28 ℃, 30 ℃, 33 ℃, 37 ℃ and cultivate, after cultivation, 8h, 16h and 24h measure respectively nutrient solution in the OD at 600nm place value.Temperature on the impact of bacterial strain L-11 growth as shown in Figure 3.Result shows: in the temperature range of experiment, thalline all has growth in various degree, when temperature is 28～30 ℃, grows best, surpass or during lower than this temperature, thalli growth speed slows down, and while cultivating at 37 ℃, bacterial classification is easily aging.Therefore, the optimum growth temp of L-11 is 28～30 ℃.3. production example

The test tube kind of gold orange II degradation bacteria is inoculated in fermention medium, and shaking culture, to logarithmic phase, is prepared inoculation seeding tank; Then above-mentioned cultured bacterial classification is inoculated into 500L seeding tank by the inoculum size of 10 ﹪, constant temperature culture is to logarithmic phase, the air flow of sterile air is 1:0.6～1.2, culture temperature is 30 ℃, and stirring velocity is 180～240r/min, and the mass content of the culture medium prescription that seeding tank is used is: glucose 0.1 ﹪, peptone 0.5 ﹪, yeast extract paste 0.25 ﹪, with sterilized water preparation, pH7.2～7.5; Seed liquor in seeding tank is produced in tank and cultivated by 10 ﹪ inoculum size accesses, produce tank used medium identical with seed tank culture base, the air flow of sterile air is 1:0.6～1.2, culture temperature is 30 ℃, stirring velocity is 180～240r/min, and whole technical process incubation time is 48～60h.After fermentation ends thalline quantity reach 1,000,000,000/more than ml.

The rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid agent.This liquid agent is microbial inoculum.

Claims (2)

1. an azoic dyestuff gold orange II degradation bacteria, is characterized in that this bacterial strain is oxidation microbacterium microbacterium oxydansbacterial strain L-11, on May 8th, 2014, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC NO.9130, Main Biological is that bacterium colony circle is opaque, cultivating 1～3 day bacterium colony is white, continuing to cultivate bacterium lives as yellow, liquid culture is yellow, cultivates 28～30 ℃ of optimum temperutures, and pH 7.2; These Pseudomonas actinomycetes.

2. the microbial inoculum that utilizes a kind of azoic dyestuff gold orange II degradation bacteria as claimed in claim 1 to produce, this microbial inoculum is to be prepared from by the following method, it is characterized in that the method comprises the following steps:

Step (1). the test tube kind of azoic dyestuff gold orange II degradation bacteria is inoculated in fermention medium, and shaking culture is to logarithmic phase;

Step (2). above-mentioned cultured bacterial classification is inoculated to the seeding tank into 500L by the inoculum size of 10 ﹪, be cultured to logarithmic phase, the culture medium prescription that seeding tank is used and mass content are: peptone 0.5 ﹪, yeast extract paste 0.25 ﹪, and with distilled water preparation, pH is 7.0;

Step (3). seed liquor is produced to tank by 10 ﹪ inoculum size accesses and cultivate, produce tank used medium identical with seed tank culture base;

Step (4). in the culturing process of seeding tank and production tank, the air flow of sterile air is 1:0.6～1.2, stirring velocity is 180～240r/min, culture temperature is 30 ℃, whole technical process incubation time is 48～60h, after fermentation ends thalline quantity reach 1,000,000,000/more than ml, the rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid agent, and this liquid agent is microbial inoculum.