CN101935626A

CN101935626A - Dimethylformamide degrading bacteria and bacterial agent produced from same

Info

Publication number: CN101935626A
Application number: CN 201010199266
Authority: CN
Inventors: 蔡天明; 陈立伟; 杨帅; 顾海莎
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2010-06-12
Filing date: 2010-06-12
Publication date: 2011-01-05
Anticipated expiration: 2030-06-12
Also published as: CN101935626B

Abstract

The invention provides dimethylformamide (DMF) degrading bacteria and a bacterial agent produced from the same, which belong to the field of biological high technology. The strain is a Gram staining reaction negative bacteria strain DMF3 and is identified as pseudomonasplecoglossicida. The strain has the main biological characteristic of G<->, an irregular rod-shaped strain body, the size of about 0.5-1.0*1.5-5.0 mu m and no stipe or mycoclena, does not produce spore, is aerobic, undergoes strict respiratory metabolism and can grow by taking DMF as a unique carbon source and nitrogen source so as to be completely mineralized for releasing ammonia nitrogen. The strain can reduce the residual quantity of the DMF with certain concentration in waste water by over 95 percent at the temperature of 30 DEG C under the pH value of 7.0 within 3 days, so that the problem of difficult treatment of the DMF in chemical production waste water is solved, the chemical oxygen demand (COD) of the DMF waste water reaches the standard and the waste water can be discharged.

Description

The microbial inoculum of a kind of dimethyl formamide degradation bacteria and production thereof

One, technical field

The invention provides the microbial inoculum of a kind of dimethyl formamide degradation bacteria and production thereof, belong to biological high-tech field, is to utilize the wastewater treatment of this chemical industry solvent commonly used of method of microorganism degraded dimethyl formamide.

Two, technical background

Dimethyl formamide (DMF) is that a kind of chemical property is stable, boiling point is high, the organic solvent of excellent property, and density is 0.94,152.8 ℃ of boiling points, zero pour-61 ℃.Except that halohydrocarbon, can dissolve each other with any ratio with water and most organic solvent, owing to its good dissolving ability, the title of " menstruum universale " is arranged.And DMF is again a kind of important agricultural chemicals medicine intermediate, the absorption agent of multiple gases: on agricultural chemicals, in order to the synthetic insecticide amidine; Pharmaceutically, in order to medicines such as synthetic Sulphadiazine Sodium, doxycycline, cortisone and vitamin B6s; In petrochemical complex, as gas absorbent, in order to separate and processed gas, as the divinyl extracting; Advantages such as usefulness DMF uses DMF good as the acrylic fibers hydrophobicity of solvent production as solvent in the production of polyamine fat synthetic leather, polyacrylonitrile reel off raw silk from cocoons technology, and spreadability is strong.Therefore in the waste water that above Chemicals production process is discharged, contain a large amount of DMF, annual only leather industry discharging contain about 100,000,000 tons of DMF waste water, environment is caused severe contamination.The DMF stable in properties, biological degradability is poor, can stimulate infringement people's eyes, and cause hepatic insufficiency, and the U.S. has determined that DMF is that human body may carcinogenic substance.DMF allows maximum concentration in China's ground water quality standard be 25mg/L.Mainly remove for middle lower concentration DMF waste water at present based on recovery and biological degradation.

Three, summary of the invention

Technical problem the objective of the invention is at practical problems in the production practice and demand, develop out a kind of dimethyl formamide degradation bacterial agent, use this microbial inoculum can make the residual quantity of DMF in the certain density DMF waste water reduce more than 95%, have wider degraded spectrum, production and use cost are lower.

Be main contents of the present invention below the technical scheme:

The invention provides a kind of degradation bacteria (Fig. 1) of dimethyl formamide, its bacterial strain is a strain gramstaining reaction negative bacterium DMF3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 18th, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number CGMCC NO.3854, through being accredited as distortion pseudomonas (Pseudomonas plecoglossicida) thalline is irregular shaft-like, about 0.5～1.0 μ m of size * 1.5～5.0 μ m, there is not stem not have sheath yet, do not produce gemma, aerobic, carry out strict respiratory metabolism, oxidase positive, the catalase positive can be sole carbon source with the dimethyl formamide, nitrogenous source is grown, and its thorough mineralising is discharged ammonia nitrogen.

The technology of using above-mentioned dimethyl formamide degradation bacteria to produce microbial inoculum is: inclined-plane kind-shake bottle a kinds-seeding tank-production jar-product (pack formulation and be liquid bacterial agent or solid absorption microbial inoculum).

Detailed implementation step of the present invention is:

1) the test tube kind with dimethyl formamide degradation bacteria DMF3 is inoculated in the LB culture media shaking vase, and shaking culture is to logarithmic phase;

2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase.The used culture medium prescription of seeding tank is: glucose 0.8%, (NH ₄) ₂

SO

₄1%, K ₂HPO ₄0.2%, MgSO ₄0.05%, NaCl 0.01%, CaCO ₃0.3%, yeast extract paste 0.02%, pH value 7.2-7.5.

3) seed liquor is produced a jar cultivation by 10% inoculum size access, it is identical with the seed tank culture base to produce the used substratum of jar.

The air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, and stirring velocity is 180-240 rev/min, and culture temperature is 30-35 ℃, and the whole process flow incubation time is 48-60 hour.Nutrient solution was microbial inoculum after fermentation was finished.

Beneficial effect

The dimethyl formamide degradation bacterial agent that uses this invention to produce has the advantage that the production use cost is low, easy to use, removal effect is good, is adapted at containing in the wastewater treatment of DMF promoting the use of.The present invention is for preserving the ecological environment, and the protection mankind's is healthy, and the cost that reduces wastewater treatment has great importance.Degradation bacteria DMF3 can grow in the minimal medium that with the dimethyl formamide is sole carbon source; Inoculum size by 2% inserts degradation bacteria DMF3 in the minimal medium that contains dimethyl formamide, under 30 ℃, pH7.0 condition, after the shaking culture 3 days, can make that certain density DMF residual quantity reduces more than 95% in the waste water, has good degradation effect (Fig. 6), degradation bacteria DMF3 has wider degraded spectrum, also can grow in organic amines such as methane amide, methylamine, dimethylamine.Greatly reduce the workload in production and the use, reduced production and use cost.

Four, description of drawings

Fig. 1 bacterial strain DMF3 bacterium colony photo

Fig. 2 degradation bacteria DMF3 growing state and relation to DMF degraded situation

Fig. 3 different vaccination amount is to the influence of DMF degradation efficiency

The initial pH of Fig. 4 is to the influence of DMF degradation efficiency

The different liquid amounts of Fig. 5 are to the influence of DMF degradation efficiency

Fig. 6 bacterial strain DMF3 is to the liquid chromatogram of DMF degraded

Five, embodiment

1 laboratory biological degradation experiment

1.1 inoculum size is to the influence of DMF degradation efficiency

Bacterial strain DMF3 the LB substratum (LB culture medium prescription g/L: yeast extract paste 5.00, peptone 10.00, NaCl 10.00, pH 7.0) in be cultured to the logarithm later stage, inoculum size by 0.5,1,2,5,10,15% inserts minimal medium (DMF 400mg/L, NaCl 1.00g/L, K respectively ₂HPO ₄2.50g/L, KH ₂PO ₄1.00g/L pH 7.0.Hereinafter the same), 30 ℃, the cultivation of 160r/min shaking table, every the content of a DMF of 12 hours mensuration, the result is as shown in Figure 3.The size of inoculum size has direct relation to the degradation efficiency of DMF, and under less than 2% inoculum size, inoculum size is big more, and the degradation efficiency of DMF is just high more; When inoculum size greater than 2% the time, it is little to DMF degraded influence to increase inoculum size again.

1.2 initial pH is to the influence of DMF degradation efficiency

Bacterial strain DMF3 is cultured to the logarithm later stage in the LB substratum, be seeded in the minimal medium of different pH by 2%, and 30 ℃, 160r/min shaking table are cultivated, and measure the wherein content of DMF behind the 72h, and the result as shown in Figure 4.Under the condition of pH5.0～9.0, bacterial strain DMF3 is bigger to the degradation rate gap of DMF; When pH is 7.0, degradation rate is almost 100%.When pH was 5.0, DMF was degraded hardly; When pH degradation rate along with the rising DMF of pH under alkaline condition descends fast.

1.3 liquid amount is to separating the influence of DMF degradation efficiency

In the triangular flask of 5 250mL, be respectively charged into 50,70,100,120, the 150mL minimal medium.Bacterial strain DMF3 cultivates the logarithm later stage in the LB substratum, and the inoculum size by 2% is inoculated in above-mentioned each substratum, 30 ℃, the cultivation of 160r/min shaking table, the degradation rate of mensuration DMF after 48 hours, result such as Fig. 5.48 hours measurement result show be equipped with 50,70,100,120, the degradation rate of DMF is respectively 86%, 80%, 70%, 66% and 63% in the triangular flask of 150mL nutrient solution, the process of bacterial strain DMF3 degraded DMF is an aerobic processes, and big air flow helps the degraded of DMF.

2 production examples

The original seed of DMF degradation bacteria DMF3 of the present invention is activated on culture dish, and measure degradation property, be inoculated on the test tube slant standby.The test tube kind is inoculated in the LB substratum 1000ml that contains 200ml and shakes in the bottle, and constant-temperature shaking culture is prepared the inoculation seeding tank to logarithmic phase.500 liters of seeding tanks, 400 liters of charging capacitys, culture medium prescription is: glucose 0.8%, (NH ₄) ₂

SO

₄1%, K ₂HPO ₄0.2%, MgSO ₄0.05%, NaCl 0.01%, CaCO ₃0.3%, yeast extract paste 0.02%, pH value 7.2-7.5.The back 121 ℃ of high pressure moist heat sterilizations that finish that feed intake, be cooled to 33 ℃ after, the above-mentioned cultured bottle bacterial classification that shakes is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 220 rev/mins, sterile air feeding amount is 1: 0.8.The seed liquor that arrives logarithmic phase is produced jar by 10% inoculum size access cultivate, it is identical with the seed tank culture base to produce the used medium component of jar.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production jar 1.1kg/cm after feeding intake ²Pressure under, 121 ℃ of high pressure moist heat sterilizations, below the sterilization postcooling to 35 ℃, logical sterile air keeps sterile state standby.A postvaccinal production jar temperature is controlled at 30-35 ℃, and the air flow of sterile air is 1 in the culturing process of production jar: 0.8-1.0, and stirring velocity is 240 rev/mins, the whole process flow incubation time is 48-60 hour.After the fermentation ends thalline quantity reach 1,000,000,000/more than the ml.

Fermentation is finished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form with plastic barrel or packing bottle or adopt peat to adsorb and be distributed into the solid fungicide formulation with packing bag.

Claims

1. dimethyl formamide degradation bacteria, it is characterized in that, this microorganism is the bacterial strain DMF3 of the distortion pseudomonas (Pseudomonas plecoglossicida) of Gram-negative, be preserved in Chinese microorganism strain on May 21st, 2010 and preserve management committee common micro-organisms center, deposit number CGMCC NO.3854, thalline is irregular shaft-like, about 0.5～1.0 μ m of size * 1.5～5.0 μ m, no stem, no sheath, do not produce gemma, aerobic, carry out strict respiratory metabolism, oxidase positive, the catalase positive can be sole carbon source with the dimethyl formamide, nitrogenous source is grown, and its thorough mineralising is discharged ammonia nitrogen.

2. degradation bacterial agent of producing with the described dimethyl formamide degradation bacteria of claim 1 is to produce by the following method:

1) the test tube kind is inoculated in the LB culture media shaking vase, shaking culture is to logarithmic phase;

2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 0.8%, (NH ₄) ₂SO ₄1%, K ₂HPO ₄0.2%, MgSO ₄0.05%, NaCl 0.01%, CaCO ₃0.3%, yeast extract paste 0.02%, pH value 7.2-7.5;

3) seed liquor is produced a jar cultivation by 10% inoculum size access, it is identical with the seed tank culture base to produce the used substratum of jar;

4) air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃, the whole process incubation time is 48-60 hour, after the fermentation ends thalline quantity reach 1,000,000,000/more than the ml, fermentation is finished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form with plastic barrel or packing bottle or adopt peat to adsorb and be distributed into the solid fungicide formulation with packing bag.