CN105176840A - Preparation method for Trichoderma harzianum degrading p-toluenesulfonic acid and industrial waste water treatment method - Google Patents
Preparation method for Trichoderma harzianum degrading p-toluenesulfonic acid and industrial waste water treatment method Download PDFInfo
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- CN105176840A CN105176840A CN201510622235.9A CN201510622235A CN105176840A CN 105176840 A CN105176840 A CN 105176840A CN 201510622235 A CN201510622235 A CN 201510622235A CN 105176840 A CN105176840 A CN 105176840A
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Abstract
The invention discloses a preparation method for Trichoderma harzianum degrading p-toluenesulfonic acid. A solid fermentation method is used for preparing the Trichoderma harzianum, and the preparation method includes the steps that straw powder is added into a fermentation vat, water and a nitrogen source are added and stirred sufficiently and evenly, and the mixture is inoculated with Trichoderma harzianum strains and make full contact with the straw powder; fermentation is carried out under the natural condition, and the temperature of a fermentation substrate is controlled not to exceed 50 DEG C. When laccase activity in the substrate is not increased, fermentation is stopped, and the obtained fermentation substrate is the Trichoderma harzianum used for treating industrial waste water. An industrial waste water treatment method with the Trichoderma harzianum includes the step that the additive amount of the Trichoderma harzianum in industrial waste water accounts for 0.1-5% of the waste water amount; the pH value of the industrial waste water is 3.0-6.0, the treatment temperature is 20 DEG C-50 DEG C, and the treatment time is 2-10 h.
Description
Technical field
The present invention relates to environment protection and biological chemical field, particularly relate to a kind of preparation of tosic acid trichoderma harzianum of degrading and the method for process trade effluent.
Background technology
Tosic acid is widely used as synthesis medicine, agricultural chemicals, the stablizer of polyreaction and the catalyzer of organic synthesis (ester class etc.); As intermediate and the resin curing agent of medicine, coating, be also used as electroplating intermediate.Tosic acid has intense stimulus effect to eyes, skin, mucous membrane and the upper respiratory tract; After suction, larynx, bronchial spasm, oedema can be caused, chemical pneumonitis or pulmonary edema, poisoning manifestations has burning sensation, coughs, pants, laryngitis, breathe hard, have a headache, nausea and vomiting.Determination of acute toxicity shows, the LD50 of tosic acid is 400mg/kg (its mouse oral), or is 2500mg/kg (rat oral).The stable chemical nature of tosic acid, can cause long-term hazards after entered environment.
Tosic acid, by trade effluent entered environment, due to features such as its toxicity is large, difficult degradations, remains in a large number in the environment, causes environmental pollution, when it enters human body by respiratory tract or human body skin, damage human body.
Tosic acid treatment process in existing trade effluent comprises: utilize the adsorption mediums such as gac, resin, sulphonated coal, realize the absorption of tosic acid in trade effluent, reach the object of industrial waste water purifying.The method treatment effect is good, can achieve qualified discharge, but adsorbs saturated gac and cannot regenerate, and can only adopt the way of burning disposal, processing costs is higher.The adsorption medium such as resin, sulphonated coal is used to there is the regeneration issues of adsorption medium and the aftertreatment problem of regenerated liquid equally.Other chemistry, photochemistry and physical chemistry means are utilized to solve the pollution of tosic acid, but this can cause secondary pollution or only make pollutent transfer to another kind from a kind of environmental system, when such as photodissociation and chemical oxidation, because luminous energy and chemical energy are to the excitation of intermediate product, also large before making intermediate product toxicity ratio process sometimes.
Summary of the invention
The present invention is directed to the problems referred to above and provide a kind of preparation of tosic acid trichoderma harzianum of degrading and the treatment process containing tosic acid trade effluent.
Being prepared as of trichoderma harzianum of a kind of tosic acid of degrading provided by the invention utilizes solid state fermentation to prepare trichoderma harziarum: first in mash-back, add straw powder, add water and nitrogenous source again, and stir, then access trichoderma harziarum bacterial classification, bacterial classification is fully contacted with straw powder; Natural condition bottom fermentation, the temperature controlling fermented substrate is no more than 50 DEG C.Stop fermentation when laccase activity no longer increases in matrix, gained fermented substrate is the trichoderma harzianum for Industrial Wastewater Treatment.
Described stalk comprises at least one in the agricultural stalks such as rice straw, wheat stalk, cotton stalk, rape stalk; Described stalk needs through pulverization process, pulverizes and needs to be the sieve plate of 1mm-2cm through aperture;
Described water consumption is 1-4 times of stalk dry weight;
Described nitrogenous source refers at least one in the inorganic nitrogens such as nitric acid ammonia, urea, amino acid, dregs of beans or organonitrogen, and various nitrogenous source is used singly or in combination, and nitrogenous source addition is to make the total C/N of system between 15-40;
Described natural condition bottom fermentation refers to yeasting temperature between 15-40 DEG C, and temperature is lower than needing heating when 15 DEG C, and temperature is higher than needing cooling when 40 DEG C;
Described control leavening temperature refers to when the temperature of the base that ferments is higher than 50 DEG C, needs turning or ventilation cooling;
The following measuring method of described laccase activity measures: 3mL reacts in cumulative volume, containing the crude enzyme liquid that 0.1mL suitably dilutes, 2.7mL0.1mol/L Acetic acid-sodium acetate damping fluid (pH4.5), add 0.2mL0.5mmol/LABTS to start reaction, under room temperature condition, under adopting UV-1700 ultraviolet-visible pectrophotometer to measure OD420, react the light absorption value change in first 3 minutes.It is 1 Ge Meihuo unit that the unit of activity (U) of enzyme is defined as per minute catalysis 1 μm of ol substrate (ABTS).Specifically see that the hair Coriolous Dersicolor (Fr.) Quel fungus of Sun Wei etc. utilizes molasses alcohol aquatic products laccase medium optimization., be published in microbiology circular 2011,38 (4): 516-522.
The method of described trichoderma harzianum process trade effluent is: the addition of described trichoderma harziarum in trade effluent is the 0.1%-5% of wastewater flow rate;
The pH that described process control comprises trade effluent is 3.0-6.0, and treatment temp is 20-50 DEG C, and the treatment time is 2-10h, determines the treatment time by the content detecting tosic acid in waste water.
Advantage of the present invention is: fully can be degraded to the toluenesulphonic acids in trade effluent by described trichoderma harziarum (Trichodermaharzianum) 1228, reach the emission request of trade effluent, effectively reduce the pollution to environment.
Accompanying drawing explanation
Fig. 1 is trichoderma harziarum of the present invention (Trichodermaharzianum) 1228 colonial morphology.
Fig. 2 is trichoderma harziarum of the present invention (Trichodermaharzianum) 1228 microscopic morphology.
Fig. 3 is the impact of pH on trichoderma harziarum degraded tosic acid degradation rate.
Fig. 4 is the impact of trichoderma harziarum bacterial classification addition on degraded tosic acid degradation rate.
Fig. 5 is the impact of temperature on trichoderma harziarum degraded tosic acid degradation rate.
Embodiment
Below by specific embodiment, the present invention is further illustrated.
The trichoderma harziarum of a kind of tosic acid of degrading of the present invention is filamentous fungal strains, called after trichoderma harziarum (Trichodermaharzianum) 1228, be preserved in the China typical culture collection center (CCTCC) being positioned at Luo Jia Shan, Wuhan City Wuhan University, preservation date is on June 27th, 2015, and deposit number is CCTCCM2015412.This bacterial strain is separation from stalk, screening obtains, through being accredited as trichoderma harziarum (Trichodermaharzianum).
The physio-biochemical characteristics of described trichoderma harziarum (Trichodermaharzianum) 1228 are:
This bacterial strain all can grow at 15-40 DEG C, and its optimum temperuture is 25-30 DEG C; When pH value 4-8, all can grow, optimum pH is 5-6; Sucrose, seminose, glucose, wood sugar, starch are conducive to the growth of mycelia, maltose and D-semi-lactosi poor; The inorganic nitrogens such as nitric acid ammonia, urea, amino acid, dregs of beans or organonitrogen are conducive to mycelial growth, and ammonium sulfate and Tryptones grow poor.
As shown in Figure 1, microscopic morphology as shown in Figure 2 for described trichoderma harziarum (Trichodermaharzianum) 1228 colonial morphology.
Being prepared as of trichoderma harzianum of a kind of tosic acid of degrading provided by the invention utilizes solid state fermentation to prepare trichoderma harziarum: first in mash-back, add straw powder, add water and nitrogenous source again, and stir, then access trichoderma harziarum bacterial classification, bacterial classification is fully contacted with straw powder; Natural condition bottom fermentation, the temperature controlling fermented substrate is no more than 50 DEG C.Stop fermentation when laccase activity no longer increases in matrix, gained fermented substrate is the trichoderma harzianum for Industrial Wastewater Treatment.
Described stalk comprises at least one in the agricultural stalks such as rice straw, wheat stalk, cotton stalk, rape stalk; Described stalk needs through pulverization process, pulverizes and needs to be the sieve plate of 1mm-2cm through aperture;
Described water consumption is 1-4 times of stalk dry weight;
Described nitrogenous source refers at least one in the inorganic nitrogens such as nitric acid ammonia, urea, amino acid, dregs of beans or organonitrogen, and various nitrogenous source is used singly or in combination, and nitrogenous source addition is to make the total C/N of system between 15-40;
Described natural condition bottom fermentation refers to yeasting temperature between 15-40 DEG C, and temperature is lower than needing heating when 15 DEG C, and temperature is higher than needing cooling when 40 DEG C;
Described control leavening temperature refers to when the temperature of the base that ferments is higher than 50 DEG C, needs turning or ventilation cooling;
The following measuring method of described laccase activity measures: 3mL reacts in cumulative volume, containing the crude enzyme liquid that 0.1mL suitably dilutes, 2.7mL0.1mol/L Acetic acid-sodium acetate damping fluid (pH4.5), add 0.2mL0.5mmol/LABTS to start reaction, under room temperature condition, under adopting UV-1700 ultraviolet-visible pectrophotometer to measure OD420, react the light absorption value change in first 3 minutes.It is 1 Ge Meihuo unit that the unit of activity (U) of enzyme is defined as per minute catalysis 1 μm of ol substrate (ABTS).Specifically see that the hair Coriolous Dersicolor (Fr.) Quel fungus of Sun Wei etc. utilizes molasses alcohol aquatic products laccase medium optimization., be published in microbiology circular 2011,38 (4): 516-522.
The method of described trichoderma harzianum process trade effluent is: the addition of described trichoderma harziarum in trade effluent is the 0.1%-5% of wastewater flow rate;
The pH that described process control comprises trade effluent is 3.0-6.0, and treatment temp is 20-50 DEG C, and the treatment time is 2-10h, determines the treatment time by the content detecting tosic acid in waste water.
The Isolation and ldentification method of trichoderma harziarum 1228 of the present invention is as follows:
Various environmental sample is dissolved in distilled water, and carries out gradient dilution, obtain the bacteria suspension of different concns; Bacteria suspension is coated on the aseptic flat board of PDA containing methyl catechol.Flat-plate inverted is placed in constant incubator cultivate, until grow the bacterium colony with red variable color circle, by bacterium colony picking purifying on PDA substratum.
After the bacterium obtained by primary dcreening operation compares, determine that bacterial strain 1228 is the strongest containing the metachrosis on the aseptic flat board of PDA of methyl catechol.
Bacterial strain, on PDA culture medium flat plate, is cultivated 3 days at 30 DEG C, the colonial morphology (as shown in Figure 1) of visual inspection bacterial strain 1228: bacterium colony is circular, and mycelia is just white cotton fiber shape, is sap green afterwards.There is pigment synthesis the local of bacterium colony, presents faint yellow.In liquid medium within, mycelia is intertwined mutually, in spherical, aobvious faint yellow.Ordinary optical microscope is observed and is shown (as shown in Figure 2), and mycelia is very thin colourless, and tool is separated, multi-branched.Crisscross, network-like.Conidiophore bears from the side shoot of mycelia, sporogenic stigma doleiform estranged or taper.Conidium mostly is spherical, sporoderm tool pustule, blue-greenish colour.
From colonial morphology and microscopic morphology, this bacterium may be Trichoderma (Trichoderma) fungi, and 18sRNA sequencing result shows, this bacterium is trichoderma harziarum (Trichodermaharzianum).
The fermentation culture concrete grammar of trichoderma harziarum 1228 of the present invention is as follows:
Get rice straw fresh then, pulverize and sieve with pulverizer, sieve diameter 1cm.Take 2000g rice straw powder in mash-back, add 60g ammonium nitrate, 5400ml water, stir, inoculating strain 1228, bacterial classification is fully fully contacted with stalk, in natural condition bottom fermentation (envrionment temperature 20-28 DEG C).After testing, to cultivate after 8 days laccase activity in system and reach 3000IU, after this no longer increase.Stop fermentation, fermented substrate is now the trichoderma harzianum of Industrial Wastewater Treatment.
The trichoderma harziarum 1228 of the present invention method of tosic acid in trade effluent of degrading is as follows:
Get tosic acid trade effluent 50ml (tosic acid content 4.8%), be loaded in 250ml Erlenmeyer flask, respectively adjust ph to 3,4,5,6,7, add 5% connect bacterium amount, constant temperature oscillation reaction 5h at 30 DEG C.When reaction system pH adjusts to about 5, the degradation rate of tosic acid is the highest, reaches 97.4% (see Fig. 3)
The present invention can regulate degradation rate to tosic acid in trade effluent by regulating trichoderma harziarum 1228 bacterial classification amount, and way is:
Get tosic acid trade effluent 50ml (tosic acid content 4.8%), be loaded in 250ml Erlenmeyer flask, adjust ph to 5, add respectively in different treatment 1%, 2%, 3%, 4%, 5% connect bacterium amount, constant temperature oscillation reaction 5h at 30 DEG C, the degradation rate of tosic acid is shown in Fig. 4.
The present invention can regulate the degradation rate in described trichoderma harziarum 1228 pairs of trade effluents by change temperature.When temperature 30 DEG C, degradation rate is best.Way is:
Get tosic acid trade effluent 50ml (tosic acid content 4.8%), be loaded in 250ml Erlenmeyer flask, adjust ph to 5, add 5% connect bacterium amount, in different treatment, control temperature constant temperature oscillation at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C reacts 5h respectively, and the degradation rate of tosic acid is shown in Fig. 5.
Claims (3)
1. the preparation method of the trichoderma harzianum of a tosic acid of degrading, it is characterized in that described preparation method prepares trichoderma harziarum for utilizing solid state fermentation: first in mash-back, add straw powder, add water and nitrogenous source again, and stir, then access trichoderma harziarum bacterial classification, bacterial classification is fully contacted with straw powder; Natural condition bottom fermentation, the temperature controlling fermented substrate is no more than 50 DEG C.Stop fermentation when laccase activity no longer increases in matrix, gained fermented substrate is the trichoderma harzianum for Industrial Wastewater Treatment;
Described stalk comprises at least one in rice straw, wheat stalk, cotton stalk, rape stalk agricultural stalk; Described stalk needs through pulverization process, pulverizes and needs to be the sieve plate of 1mm-2cm through aperture;
Described water consumption is 1-4 times of stalk dry weight;
Described nitrogenous source refers at least one in the inorganic nitrogens such as nitric acid ammonia, urea, amino acid, dregs of beans or organonitrogen, and various nitrogenous source is used singly or in combination, and nitrogenous source addition is to make the total C/N of system between 15-40;
Described natural condition bottom fermentation refers to yeasting temperature between 15-40 DEG C, and temperature is lower than needing heating when 15 DEG C, and temperature is higher than needing cooling when 40 DEG C;
Described control leavening temperature refers to when the temperature of the base that ferments is higher than 50 DEG C, needs turning or ventilation cooling.
2. the preparation method of the trichoderma harzianum of a kind of tosic acid of degrading according to claim 1, it is characterized in that the following measuring method of described laccase activity measures: 3mL reacts in cumulative volume, containing the crude enzyme liquid that 0.1mL suitably dilutes, 2.7mL0.1mol/L Acetic acid-sodium acetate damping fluid (pH4.5), add 0.2mL0.5mmol/LABTS to start reaction, under room temperature condition, under adopting UV-1700 ultraviolet-visible pectrophotometer to measure OD420, react the light absorption value change in first 3 minutes.It is 1 Ge Meihuo unit that the unit of activity (U) of enzyme is defined as per minute catalysis 1 μm of ol substrate (ABTS).
3. the method for the trichoderma harzianum process trade effluent of a kind of tosic acid of degrading according to claim 1 is: the addition of described trichoderma harziarum in trade effluent is the 0.1%-5% of wastewater flow rate; The pH of described trade effluent is 3.0-6.0, and treatment temp is 20-50 DEG C, and the treatment time is 2-10h, determines the treatment time by the content detecting tosic acid in waste water.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400700A (en) * | 2016-01-26 | 2016-03-16 | 嘉兴学院 | Preparation method of methyl p-tolyl sulfone degrading bacterium trichoderma asperellum and method for treatment of industrial wastewater |
| CN105586279A (en) * | 2016-03-16 | 2016-05-18 | 南京农业大学 | Method for preparing trichoderma solid spawn through trichoderma direct fermentation crop straw and prepared product |
| CN106085871A (en) * | 2016-06-18 | 2016-11-09 | 南京农业大学 | A kind of be raw material production Trichoderma spp. solid spawn with amino acid hydrolyticsolution and rice straw method |
| CN110845019A (en) * | 2019-11-07 | 2020-02-28 | 桂林理工大学 | Method for degrading diethylstilbestrol by using coriolus versicolor crude enzyme solution |
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2015
- 2015-09-25 CN CN201510622235.9A patent/CN105176840A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400700A (en) * | 2016-01-26 | 2016-03-16 | 嘉兴学院 | Preparation method of methyl p-tolyl sulfone degrading bacterium trichoderma asperellum and method for treatment of industrial wastewater |
| CN105586279A (en) * | 2016-03-16 | 2016-05-18 | 南京农业大学 | Method for preparing trichoderma solid spawn through trichoderma direct fermentation crop straw and prepared product |
| CN105586279B (en) * | 2016-03-16 | 2016-12-28 | 南京农业大学 | A kind of Trichoderma spp. direct fermentation crop material prepares the method for Trichoderma spp. solid spawn and the product of preparation |
| CN106085871A (en) * | 2016-06-18 | 2016-11-09 | 南京农业大学 | A kind of be raw material production Trichoderma spp. solid spawn with amino acid hydrolyticsolution and rice straw method |
| CN110845019A (en) * | 2019-11-07 | 2020-02-28 | 桂林理工大学 | Method for degrading diethylstilbestrol by using coriolus versicolor crude enzyme solution |
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Application publication date: 20151223 |