CN1294088C - Biological method for treating synthetic pharmaceutical waste water by specific strain - Google Patents

Abstract

The present invention relates to a method for degrading and treating synthetic pharmaceutical waste water by specific strains, which uses genetic engineering bacteria to treat the pharmaceutical waste water by the protoplast fusion of three parent strain bacteria which are a white-rot fungi (parent strain 1), an Alfur bacteria XZ1(parent strain 2)and a saccharomyces cerevisiae (parent strain 3). In the method, firstly, the pH of the waste water is regulated to be 6.8 to 8.0 in a regulation tank; afterwards, the waste water is aerated and deposited in a reactor, and sludge in a sludge tank back flows to an aeration reaction tank with the treating temperature of 30(+/-)2 DEG C; oxygen dissolved in the reactor is higher than or equal to 2 mg/L, and the bacterium concentration is 2 g/L to 10 g/L. The degradation ratio q <max> with the maximum ratio of the present invention for degrading the synthetic pharmaceutical waste water is 11.188 d<-1>. The present invention has the advantages of high degradation, flocculation and adaptability, increases the biological load efficiency by more than 56%, saves the construction cost of reactor volume by 68%, generates no new genes in protoplast fusion, and has no gene pollution problem by using NJU-Xhhh1 specific strains.

Description

A Biotechnological Approach to Treating Synthetic Pharmaceutical Wastewater with Engineered Strains

technical field

The invention relates to a biotechnological method for processing synthetic pharmaceutical wastewater by engineering microorganism Xhhh strain.

Background technique

There are many kinds of wastewater treatment engineering strains in the world, but there is no patent on the application of protoplast fusion engineering strains to treat synthetic pharmaceutical wastewater. Typical examples are EM in Japan, and HIMP (High-Intelligence Microorganism Preparation) from PARADISF in the United States. However, the application of protoplast fusion engineering strains at home and abroad to treat synthetic pharmaceutical wastewater is still blank. For example, a company synthesizes and produces neuromodulatory drugs, and the wastewater contains organic chlorine and other organic pollutants such as benzene rings and heterocyclics, among which persistent organic pollutants POPs (persistent organic pollutants) and environmental hormones EH (environmental hormone) are polluted There are as many as 16 kinds or more. They are difficult to be quickly degraded by indigenous microorganisms. They have molecular genotoxicity that can cause cancer and reduce the quantity and quality of sperm in humans, and have molecular ecotoxicity that can potentially destroy biodiversity in the environment. Existing strains cannot effectively and stably treat high-concentration pharmaceutical organic wastewater.

Contents of the invention

The purpose of the invention is to provide a method for efficiently treating synthetic pharmaceutical wastewater with protoplast fusion engineering strains. In particular, the original genetically engineered bacteria NJU-Xhhh1 constructed by the present invention is used as the starting strain, which has high degradability of white rot fungi, high adaptability of indigenous bacteria XZ1, and high flocculation of Saccharomyces cerevisiae, which is beneficial to efficient treatment of synthetic pharmaceuticals Organic waste water.

The object of the present invention is achieved in this way: the original engineering strain is used to process the biotechnology of synthetic pharmaceutical organic wastewater. (1). Technical idea: The original genetically engineered bacteria NJU-Xhhh1 constructed by the present invention is used as the starting strain, and the strain has high degradability of white rot fungi, high adaptability of indigenous bacteria XZ1, and high flocculation of Saccharomyces cerevisiae , which is conducive to the efficient treatment of synthetic pharmaceutical organic wastewater. Supporting related application technologies are original and durable at home and abroad.

(2).Technical composition:

Starting strain NJU-Xhhh1 engineering bacteria: it is the original scientific research achievement of the present invention. The NJU-Xhhh1 engineering bacteria is fused with the protoplasts of three parent strains: white rot fungus (parent strain 1), indigenous bacteria XZ1 (parent strain 2), and Saccharomyces cerevisiae (parent strain 3). Recombine and integrate to construct the obtained genetically engineered bacteria, which is the No. 1087 microorganism of the China Center for Microorganisms General Microorganisms. The NJU-Xhhh1 engineering bacteria combines the three high advantages of high degradability, high adaptability and high flocculation of the three parent strains, which is conducive to the efficient treatment of pharmaceutical organic wastewater. It is internationally recognized that the new strain constructed by protoplast fusion does not have the problem of genetic pollution, so the NJU-Xhhh1 engineering bacterial agent does not have the problem of genetic pollution.

The method for treating pharmaceutical wastewater with the genetically engineered bacteria is as follows: the wastewater is first adjusted to an acid-base condition in a regulating tank, and the pH value is 6.8-8.0; then aeration and precipitation are carried out in the reactor, and the sludge in the sludge tank is refluxed to Aeration reaction tank, its treatment process parameters are: treatment temperature, 30±2°C; dissolved oxygen in the reactor ≥ 2mg/L; bacteria concentration, 2-10g/L.

The NJU-Xhhh1 engineered bacteria is formed by the fusion of parent strain 1 white rot fungus (Phanerochaete chrysosporium), parent strain 2 indigenous bacteria XZ1 (Bacillus), and parent strain 3 Saccharomyces cerevisiae. Preparation of protoplasts first: remove the cell wall of fungi with helicase or remove the cell wall of bacteria with lysozyme, generate protoplasts, collect the protoplasts by centrifugation, wash with buffer, and then carry out the second fusion, the first fusion : Equal amounts of parent strain 1 white-rot fungal cells and parent strain 2 native bacterial YZ1 cell protoplasts were mixed and fused in an induction solution prepared with polyethylene glycol (PEG, MW=6000), CaCl ₂ , and sucrose, After centrifugation, the fusion product was collected, and the hyperosmotic buffer was centrifuged and washed, and then spread on the three solid identification media of SIM1, SIM2, and SIM3, and cultured at 30°C for 7 days, and the colonies that could grow on the three media at the same time, Xhh is the product of the first parental cross-kingdom fusion; while parental strain 1 white-rot fungal cells and parental strain 2 indigenous bacterial XZ1 cells can only grow on SIM1 and SIM2 respectively; the second fusion: the same amount obtained last time The protoplasts of Xhh and parent strain 3 Saccharomyces cerevisiae cells were mixed and fused in the induction solution prepared with polyethylene glycol (PEG, MW=6000), CaCl ₂ and sucrose, and the fusion product was collected after centrifugation, containing high sucrose After centrifugation and washing with osmosis buffer, spread on three solid identification media of SIM1, SIM2, and SIM3 respectively, and the colonies that can grow on the three media of SIM1, SIM2, and SIM3 at the same time are the product of three-parent cross-border fusion NJU -Xhhh1 or Xhhh.

Among them: white rot fungus PC [eukaryotic cell], indigenous fungus XZ1 [prokaryotic cell], Saccharomyces cerevisiae SC [eukaryotic cell].

SIM1=SM+100u streptomycin/ml (streptomycin, Sm);

SIM2=SM+100u nystatin/ml (nystatin, Nt);

SIM3=SM+100u streptomycin/ml+100u nystatin/ml (Sm+Nt).

Liquid medium for preparing NJU-Xhhh1 engineering bacterial agent: in 1000ml: K ₂ HPO ₄ 3g; KH ₂ PO ₄ 1g; NH ₄ NO ₃ 0.5g; Na ₂ SO ₄ 0.1g; MgSO ₄ 7H ₂ O 10mg; MnSO ₄ 4H ₂ O 1mg; CaCl ₂ 0.5mg; FeSO ₄ 7H ₂ O 1mg; CH ₃ COONa 5g; Yeast extract 5g; Peptone 10g; Glucose 10g; , Moist heat sterilization for 20 minutes. Optimum culture temperature: 35±2℃; optimum pH: 7.0.

Fermentation process for preparing NJU-Xhhh1 engineering bacterial agent:

Fermentation equipment: fully automatic bioreactor;

Fermentation process: continuous and stable fermentation system; fermentation temperature, 33±2°C; pH value, 7.0±1; dissolved oxygen in the reactor, ≥2mg/L;, mineral salt flow rate, 0.001-0.005V/(V.d); cells Concentration, 2-10g/L; cultivated in culture medium containing C, P, and N.

Table 1. NJU-Xhhh1 Engineering Bacteria Fermentation Process Parameters Process parameters value Process parameters value T, fermentation temperature, °C, tank pressure, (1 atmosphere = 10 ⁵ Pa), PaFa, clean air supply volume, m ³ /kgCODRr, stirring speed, rpmPHDO, dissolved oxygen in the reactor, mg/LVa, reaction solution Loading capacity, %So, C source concentration in culture solution, gCOD/LSn, N source concentration in culture solution, gTN/L 30±25×10 ⁵ <75≤10007.0±1≥280≤8.480.50 Sp, P source concentration in culture solution, gTP/LSf, C/N/P culture solution flow rate, V/(Vd)Mr, mineral salt flow rate, V/(Vd)X, cell concentration, g/LHRT, reaction solution fermentation Time, hFr, flow rate μ of silicone oil defoamer, specific growth rate, mg/mgL h ^-1 q, specific utilization rate of culture medium, h ^-1 Yo, bacterial cell yield coefficient, % 0.100.330.0034.998Sf<0.1270.21159

Beneficial effects of the invention:

(1) The production cost of NJU-Xhhh1 engineering bacterial agent is low, only about 500 yuan/ton, and the price can be much lower than imported bacterial agents;

(2) The NJU-Xhhh1 engineering bacterial agent has the advantages of the three highs of the three-parent strain; it improves the treatment efficiency and saves the cost by more than 50%;

(3) NJU-Xhhh1 engineering bacterial agent can treat both toxic organic wastewater and conventional organic wastewater;

(4) NJU-Xhhh1 engineering bacteria agent uses protoplast fusion engineering bacteria, does not produce new genes, and has no gene pollution problem.

In a word, the bacterial agent of the present invention improves the waste water degradation efficiency and saves operating costs by more than 50% compared with the native bacteria. The invention is an original engineering bacterium using protoplast fusion, and is the first example of preparing and treating synthetic pharmaceutical wastewater engineering bacterium. Especially the application of internationally original engineering bacteria, the prepared engineering bacteria agent has the advantage of domestic promotion in terms of cost and selling price. Degradation of synthetic pharmaceutical wastewater has the advantages of high degradation, high adaptability, high flocculation, and good stability and adaptability.

Description of drawings

Fig. 1 is the schematic diagram of the technical process of synthetic pharmaceutical wastewater treatment by engineering strain NJU-Xhhh1 of the present invention

Detailed ways

Refer to Figure 1 for the fermentation process flow of the NJU-Xhhh1 engineering bacterial agent preparation technology: especially aeration is performed through multi-stage aeration tanks (three stages in the figure), and the sedimentation tank returns to the first aeration tank. After two to three stages of reactor aeration.

Example: Preparation of NJU-Xhhh1 Engineering Bacteria and Its Degradation Synthesis of Pharmaceutical Wastewater

(1) Preservation formula of NJU-Xhhh1 engineering bacterial agent preparation (4°C)

Formula 1: NJU-Xhhh1 engineering bacterial agent fermentation broth, without any other ingredients;

Formula 2: NJU-Xhhh1 engineering bacterial agent fermentation broth + antioxidant mercaptoethanol (SH); (final concentration 0.2‰);

Formula 3: NJU-Xhhh1 engineering bacterial agent fermentation broth + Fe ³⁺ , (final concentration 0.14%);

Formula 4: NJU-Xhhh1 engineering bacterial agent fermentation broth + Fe3 ⁺ -mercaptoethanol (SH); (final concentration 0.2‰);

(2) Summary of preparation examples of NJU-Xhhh1 engineering bacterial agent

The fermentation process control condition of preparing engineering bacterial agent is: DO (dissolved oxygen)=2mg/L; pH=7.0; T=35 ℃; Sf (liquid medium flow rate)=0.33 (V/V); Stirring speed: 1000rpm; Silicone oil removes foam; Mineral salt flow rate: 0.003 (V/V); The best condition for storing NJU-Xhhh1 engineering bacterial agent is: 4°C storage, engineering bacteria fermentation broth + mercaptoethanol (SH);

(3) Background of water quality parameters of synthetic pharmaceutical wastewater degraded by NJU-Xhhh1 engineering strain parameter name parameter value parameter name parameter value parameter name parameter value COD _cr (mg/L)BOD ₅ (mg/L)PHT(℃)TP(mg/L)TN(mg/L)Al(mg/L)Ba(mg/L)Be(mg/L) 4123276.5-8.0250.603.001.000.094＜0.002 Ca(mg/L)Cd(mg/L)Co(mg/L)Cr(mg/L)Cu(mg/L)Fe(mg/L)K(mg/L)Mg(mg/L)Mn( mg/L) 136<0.0030.006<0.0080.2101.24011320.087 Na(mg/L)Pb(mg/L)Se(mg/L)Si(mg/L)Sr(mg/L)Ti(mg/L)V(mg/L)Zn(mg/L)PoPs types 202<0.050<0.0500.2400.5200.015<0.0030.24016 kinds or more

(4) Kinetic parameters of NJU-Xhhh1 engineering strain (agent) to degrade synthetic pharmaceutical wastewater: serial number Kinetic parameters XZ parent strain PC parent strain SC parent strain Xhhh 123456 Maximum specific degradation rate q _max (d ^-1 )q _max /2 constant K _Sq (mg/L) maximum specific growth rate μ _max (d ^-1 ) μ _max /2 constant K _sμ (mg/L) theoretical yield coefficient Y _T (%) cell attenuation coefficient K _d (d ^-1 ) 3.8060.5172.1970.5450.5690.012 11.9980.9000.3570.5710.0470.026 2.5340.7120.2201.0350.0990.024 11.1880.9782.4691.0910.2060.019

(5) The biotechnological optimization results of NJU-Xhhh1 engineering strain (agent) degrading and synthesizing pharmaceutical wastewater: the main control parameters are: the sludge return concentration is controlled at 5-5.5 (kg/m ³ ); the hydraulic retention time is 0.6-0.65d ;The sludge residence time is 20-21d; serial number 12 Parameter name and unit influent flow Qo(m ³ /d) influent So(COD _cr , kg/m ³ ) Engineering strain Xhhh optimization technology 6000.412 Indigenous bacteria XZ original technology 6000.412 34567891011121314151617181920 Effluent Se (COD _cr , kg/m ³ ) Effluent suspended solids Xe (kg/m ³ ) Aeration tank biomass X (kg/m ³ ) Hydraulic retention time θ(d) Sludge retention time θ _c (l/μ , d) return sludge concentration Xr (kg/m ³ ) return sludge flow Qr (m ³ /d) sludge discharge concentration Xs (kg/m ³ ) sludge discharge flow Qs (m ³ /d) observed yield Coefficient Yobs (%) Theoretical yield coefficient Y _T (%) Endogenous respiration rate of sludge Kd(d ^-1 ) Specific growth rate μ(d ^-1 ) Specific degradation rate q(d ^-1 ) Total biomass of aeration reaction Wa (kg) total pollutant removal U (Kg/d) sludge discharge dry weight Ws (Kg/d) required aeration tank volume V (m ³ ) 0.0250.00022.7360.63721.885.3956005.39510.0000.20010.20580.01920.04570.22221045232.247.8Vmin＝382 0.1350.07003.0610.92718.666.0366006.03615.1140.54860.56890.00180.05360.09771702166.291.2V＝556

Save 68% of reactor volume construction cost; increase bioburden efficiency by 56%.

Claims (9)

1. A method for degrading and treating synthetic pharmaceutical wastewater by engineering bacterial agents, characterized in that it is fused with the protoplasts of the three parent strains of parent strain 1 white rot fungus, parent strain 2 indigenous bacteria, and parent strain 3 Saccharomyces cerevisiae. Recombination and integration in the same cell to construct the obtained genetically engineered bacteria; then the method of using the genetically engineered bacteria to treat pharmaceutical wastewater is: the wastewater is first adjusted to acid and alkali through the adjustment tank, the pH value is 6.8-8.0; then in the reaction Aeration and sedimentation in the reactor, and returning the sludge in the sludge tank to the aeration reaction tank, the treatment process parameters are: treatment temperature, 30±2°C; dissolved oxygen in the reactor ≥ 2mg/L; bacterial concentration, 2-10g/L. 2. The preparation method of the engineering bacterial agent according to claim 1 is characterized in that: it is processed in a nutrient element containing P and N, and the mineral salts of the P and N nutrient elements include Na ₂ SO ₄ , MgSO ₄ ·7H ₂ O, MnSO ₄ .4H ₂ O, CaCl ₂ , FeSO ₄ .7H ₂ O. 3. The preparation method of the engineering bacterial agent according to claim 1, characterized in that the composition of the liquid medium for preparing the genetically engineered bacteria is: in 1000ml: K ₂ HPO ₄ 3g; KH ₂ PO ₄ 1g; NH ₄ NO ₃ 0.5g; Na ₂ SO ₄ 0.1g; MgSO ₄ 7H ₂ O; 10mg; MnSO ₄ 4H ₂ O 1mg; CaCl ₂ 0.5mg; FeSO ₄ 7H ₂ O 1mg; CH ₃ COONa 5g; yeast extract 5g; peptone 10g; glucose 10g; 200g potato leaching juice, adjust the pH to 7.0; the culture method is to inoculate and ferment after sterilizing at 121°C, 103kPa, and damp heat for 20min, culture temperature: 33-37°C; pH: 7.0, ferment for 10 -25 hours. 4, by the preparation method of the described engineering bacterial agent of claim 1, it is characterized in that described genetic engineering bacterial agent preparation is preserved in antioxidant mercaptoethanol final concentration 0.1-0.3‰+Fe ³⁺ final concentration 0.1-0.14% Engineering bacteria agent fermentation broth. 5. The method for degrading and treating synthetic pharmaceutical wastewater by the engineering bacteria agent as claimed in claim 1, characterized in that the sludge return concentration is controlled to be 5-5.5kg/m ³ ; the hydraulic retention time is 0.6-0.65d; the sludge retention time It is 20-22d; the return flow control is 100%. 6. The method for degrading and treating synthetic pharmaceutical wastewater with the engineering bacterial agent as claimed in claim 1, characterized in that the concentration of sludge return flow is controlled to 5.395kg/m ³ . 7. The method for degrading and treating synthetic pharmaceutical wastewater by the engineering bacterial agent as claimed in claim 1, characterized in that the hydraulic retention time is 0.6-0.65d. 8. The method for degrading and treating synthetic pharmaceutical wastewater by the engineering bacterial agent as claimed in claim 1, characterized in that the sludge residence time is 20-22d. 9. The method for degrading and treating synthetic pharmaceutical wastewater with the engineering bacterial agent as claimed in claim 1, which is characterized in that the reactor is aerated two to three times.