CN103466806A - Process technology for degrading gallic acid in waste water by employing aspergillus oryzae - Google Patents
Process technology for degrading gallic acid in waste water by employing aspergillus oryzae Download PDFInfo
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Abstract
The invention discloses a process technology for degrading gallic acid in waste water by employing aspergillus oryzae, and relates to the field of bioengineering. Namely, the process technology comprises the following steps: with the aspergillus oryzae as an original strain, transferring a bacteria solution to the waste water containing the gallic acid by liquid shake-flask culture and liquid strain enlargement cultivation, and supplying certain nutrient substances; and carrying out fermented cultivation. By adopting the technology, the removal rate of the gallic acid can be up to 60-100%; meanwhile, thalli and active molecules used as feed additives can be obtained.
Description
Technical field
The present invention relates to technical field of bioengineering, refer in particular to the waste water that utilizes the aspergillus oryzae degraded to contain gallic acid.Raw wastewater is concentrated or is diluted to gallic acid content after 1～50 grams per liter, supplemental medium composition, the gallic acid in access aspergillus oryzae bacterial classification degrading waste water.After the aspergillus oryzae degraded, the clearance of gallic acid can reach 60～100%, BOD clearance and reach 90～99%, and the waste water after simultaneously degrading can extract mycelium and activeconstituents is used as fodder additives.
Background technology
(the Dissolved organic matter of dissolved organic matter in natural water body, DOM) main component is natural organic acids (Natural organic acids, NOA), and 15% one 30% be the hydrophilic small molecules organic acid in natural organic acids, gallic acid is as the ubiquitous hydrophilic small molecules organic acid of nature, can bring following problem in the drink water purifying process: the colourity that 1) affects water, mouthfeel and smell: 2) form disinfection byproduct (DBP) (Disinfection byproducts in the chlorine disinfectant process, be called for short DPBs), as trichloromethane, the halogen acetic acid class, they can be to organism especially mankind liver in water body and after chloropexia, kidney and central nervous system cause damage, have carcinogenic, teratogenesis, mutagenesis, 3) can easily cause the pollution of film in the advanced treatment process of follow-up water.Gallic acid is also important source material used in food-processing, daily use chemicals, medicine production, in production process, institute's effluent contains a large amount of gallic acids, it can decompose produce organic acid and biogas etc. in water body, consumes oxygen in water, causes the hydrobiont anoxic such as fishes and shrimps and death; Cause that water body COD raises etc., bring thus a series of water ecological environment problem.Therefore in the efficient degradation water body, gallic acid becomes an importance of water pollution control.
At present about the processing patent that contains gallic acid waste water, there are several, but be mainly the methods such as washing or alkaline extraction, as patent method of recovering and treating gallic acid sludge (application number 200610086067.7), protected waste residue twice washing under 90 ~ 100 degree, the method for twice squeezing reclaims the method for gallic acid; Patent technology for recovering mother solution of gallic acid (patent No. 200910226622.5) has been protected and has been utilized the acid of alkaline extraction gallic acid, then regulates the technique that the pH8.0-9.0 precipitation obtains the gallic acid crude product; Patent gallic acid production wastewater, waste residue, useless carbon recycling new technology (number of patent application 201210469098.6) disclose the waste residue of producing gallic acid, useless charcoal adopts extrusion process of adverse current secondary washing to reclaim gallic acid technique; A kind of method (application number 2010136553.1) and article " Fenton reaction-neutralization-biological process is processed the gallic acid crude wastewater " (magazine " water technology " 2010 of processing the crude wastewater of gallic acid of patent, the 9th phase of 36 volumes, pp106-109), disclose the gallic acid crude wastewater through catalyzed oxidation, regulate pH precipitation, charcoal absorption, finally by crossing the method for diluting Air Exposure, these methods all exist to be processed not exclusively, or dilutes with a large amount of water.
The inventor is through deep research, find out a kind of with aspergillus oryzae (
aspergillus oryzae) as starting strain, the method that degraded contains gallic acid waste water.The method be different from method in the past be used bacterial classification by united States food and drug administration (FDA) and U.S. feed public determine association (AAFCO), the identification of China Ministry of Agriculture can the Direct-fed animal the aspergillus oryzae bacterial classification of feed level microbe additive, gallic acid in degrading waste water, the activeconstituents that obtains tropina simultaneously and contain the fermented liquids such as one or more zymins (saccharifying enzyme, proteolytic enzyme, phytase, lipase).These thalline and activeconstituents can be used as fodder additives.
Summary of the invention
The present invention is to provide the biotechnology of gallic acid in the aspergillus oryzae degrading waste water.Waste water is after the aspergillus oryzae degraded, and its gallic acid clearance can arrive 60 ~ 100%.
A kind of aspergillus oryzae degraded of the present invention gallic acid Technology, according to following step, carry out: take aspergillus oryzae as starting strain, carry out test tube enlarged culturing, liquid shaking bottle cultivation, thalline fluid enlargement culture, centrifugal collection thalline, then by gallic acid in fermentative degradation waste water.Waste water after degraded is through the aspergillus oryzae filament of centrifugal acquisition, and filtrate obtains multiple bioactive molecules through molecular retention, and these mycelium and bioactive molecules can be used in fodder additives.
The present invention's bacterial classification used is any bacterial classification of aspergillus oryzae, as the bacterial classification of Chinese common micro-organisms culture presevation administrative center (CCGMC), Chinese agriculture microbial strains preservation administrative center (ACCC), Chinese industrial microbial strains preservation administrative center (CICC), Chinese medicine microbial strains preservation administrative center (CMCC), national veterinary microorganism DSMZ (CVCC) and microbiotic bacterial classification preservation pipe reason center etc. preservation.
The applicable waste water of the present invention is any one waste water that contains gallic acid, extracts the waste liquid of gallic acid as the waste liquid of prodn. of gallic acid by fermentation process, from plant or take the waste liquid of gallic acid as synthetic other products of raw material.
Test tube enlarged culturing base in wherein said test tube enlarged culturing is potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages.
During wherein said liquid shaking bottle is cultivated and liquid shaking bottle substratum and liquid spawn culture medium in the thalline fluid enlargement culture be: glucose 5～30 grams per liters, peptone 0～5 grams per liter, yeast extract paste 0～5 grams per liter, sal epsom 0.2～1 grams per liter, tween 80 0.5～10 grams per liter, potassium primary phosphate 2～9 grams per liters, pH5～8,120～140 ℃ sterilizing 20～40 minutes; Wherein said shake-flask culture processing condition are to inoculate a ring aspergillus oryzae test tube slant spore (1 * 10
6) in the 250mL triangular flask that 40～120 mL substratum are housed, at rotating speed: 150 rev/mins, 25～35 ℃, cultivate 24～72 hours; Wherein said first order seed culture process is, by 1～20%(volume ratio) inoculum size the shake-flask culture seed liquor is inoculated in the first order seed substratum, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 18～72 hours;
The waster water process that degraded of the present invention contains gallic acid is that the waste water that contains gallic acid makes gallic acid concentration reach 1～50 grams per liter waste water 1000L, 5～30 kilograms of glucose, 0.5～2 kilogram of soybean cake powder through concentrated or dilution; Copper sulfate 5～15 grams, ferrous sulfate 5～15 grams, manganous sulfate 5～20 grams, each composition of this substratum can amplify in this ratio; Wherein said degradation technique is: by 2～20%(seed liquor volume/wastewater volume) inoculation level liquid bacterial classification, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 36～168 hours.
After using this processing wastewater degraded, the gallic acid clearance can reach 60-100%.
According to fermentating culturing process of the present invention, in conjunction with the ABC of this area, can be according to need of production, increase even level Four seeding tank of secondary, three grades, further expand the scale of production, to carry out suitability for industrialized production.Secondary, the three grades substratum that even the level Four seeding tank adopts are identical with the first order seed substratum with the shake-flask culture base, and inoculum size is 5～20%, and culture condition is same as liquid spawn enlarged culturing condition,
Another object of the present invention is to provide activeconstituents that in a kind of aspergillus oryzae of usining degrading waste water and fermented liquid, molecular retention obtains as fodder additives, it is characterized in that the distillery waste of above-mentioned aspergillus oryzae degraded, centrifugal 5～20 minutes of 3000～6000rpm, obtain mycelium, mycelium is dried, and preserves.Filtrate, hold back concentrated 50～100 times by the film of molecular weight 5000～10000, and-20 ℃ of lyophilizes, obtain macromolecular activeconstituents.Due to many documents, (punishment carrys out monarch, common mycology, 1999) report, the multiple enzymes such as aspergillus oryzae can extracellular proteinase, amylase, lipase, polygalacturonase, phytase, saccharifying enzyme, therefore infer that this activeconstituents contains following one or more enzymes: as saccharifying enzyme, proteolytic enzyme, phytase, lipase.～100 times ,-20 ℃ of lyophilizes, every liter of waste liquid can obtain 2-5 gram mycelium and bioactive molecule.These mycelium and bioactive molecule can be used as fodder additives.
Embodiment
According to the gallic acid measuring method of this process using, be: precision takes gallic acid reference substance 10.3 mg, is placed in 100 mL beakers, dissolves with hplc grade methanol, and is settled in the 100mL volumetric flask, is mixed with the standard reserving solution of 10.3mg/mL.Face the used time with 0.45 μ m filtering with microporous membrane, controlling sample size is 2,4,6,8, and 10 μ l utilize high performance liquid chromatograph (HIMADZU (Shimadzu) LC-20AT) to measure gallic acid content.Chromatographic column is Alltima
tMc18 post (4.6 mm * 250 mm, 5 μ m).Condition determination is moving phase: methyl alcohol: 0.1% phosphoric acid solution=10:90; Column temperature: 30 ℃, flow velocity: 0.8 mL/min, detect wavelength: 270 nm.Take sample size as X-coordinate (X), and the corresponding peak area of take carries out linear regression as ordinate zou (Y), tries to achieve the equation of linear regression of gallic acid concentration and peak area.The supernatant liquor of drawing sample liquid (before degraded and in degradation process and after degraded) is a small amount of, with 0.45 μ m filtering with microporous membrane, measure respectively peak area by above-mentioned chromatographic condition, adopt the regression equation calculation gallic acid content of external standard peak area according to above-mentioned gallic acid concentration of trying to achieve and peak area.
Gallic acid clearance Re calculates by following formula (1):
In formula,
r t clearance for t time gallic acid;
c 0 (mg/L) be the gallic acid concentration of 0 hour;
c t (mg/L) for adsorbing the concentration of t hour gallic acid.
In order further to set forth related material and the technique of technical scheme of the present invention, provided following examples, but these embodiment do not limit the scope of the invention in any form.
Embodiment 1
1. the making of test tube slant bacterial classification
Access one ring aspergillus oryzae in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration (
aspergillus oryzae) bacterial classification (Chinese common micro-organisms culture presevation administrative center CCGMC3.5232), 26 ℃, incubation time 50 hours; 4 ℃ save backup.
2. liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 5 grams, peptone 0 gram, yeast extract paste 5 grams, sal epsom 0.2 gram, tween 80 0.5 gram, potassium primary phosphate 2 grams, water 1000 mL, pH 5, packing 250mL triangular flask, every bottle of 50 grams, totally 20 bottles, 120 ℃ of sterilizings 40 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 4 ℃ of preservations of a ring, at 25 ℃, 150 rev/mins, cultivates 72 hours;
3. the making of level liquid bacterial classification
Accurately take glucose 50 grams, peptone 0 gram, yeast extract paste 50 grams, sal epsom 2 grams, tween 80 5 grams, potassium primary phosphate 20 grams, water 10L, be loaded in the seeding tank of 15L, 120 ℃ of sterilizings 20 minutes; After sterilizing is cooling, 25 ℃ of temperature, 50 rev/mins of mixing speed, 0.2: 1 volume/volume of air flow/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 72 hours;
4. wastewater degradation
Take Turkey-galls as raw material, adopt the waste liquid of High Temperature High Pressure and alkaline extraction gallic acid through concentrated, the waste liquid 1000L that gallic acid concentration is 50 grams/L, 5 kilograms of glucose, 0.5 kilogram of soybean cake powder; Copper sulfate 5 grams, ferrous sulfate 5 grams, manganous sulfate 5 grams; Access 2%(seed liquor volume/wastewater volume) inoculation level liquid bacterial classification, 25 ℃ of temperature, 60 rev/mins of mixing speed, air flow 0.3:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate after 44 hours, the gallic acid clearance reaches 60.2%.
5. thalline and active substance extract
The gallic acid waste water of above-mentioned aspergillus oryzae degraded, centrifugal 6 minutes of 3000rpm, obtain mycelium, and mycelium is dried, and preserves.Filtrate, hold back concentrated 50 times by the film of molecular weight 5000, and-20 ℃ of lyophilizes, obtain macromolecular activeconstituents.This activeconstituents contains following one or more enzymes: as saccharifying enzyme, proteolytic enzyme, phytase, lipase.Every liter of waste liquid can obtain activeconstituents outside thalline and born of the same parents and weigh 4.9 kilograms altogether.
Embodiment 2
1. the making of test tube slant bacterial classification
Access one ring aspergillus oryzae in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration (
aspergillus oryzae) bacterial classification (Chinese agriculture microbial strains preservation administrative center ACCC30155), 30 ℃, incubation time 100 hours; 7 ℃ save backup.
2. liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 150 grams, peptone 25 grams, yeast extract paste 25 grams,, sal epsom 0.4 gram, tween 80 4 grams, potassium primary phosphate 5 grams, water 10 L, pH 6, packing 250mL triangular flask, every bottle of 100 grams, totally 100 bottles, 130 ℃ of sterilizings 30 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 7 ℃ of preservations of a ring, at 30 ℃, 150 rev/mins, cultivates 50 hours;
3. the making of level liquid bacterial classification
Accurately take glucose 2000 grams, peptone 250 grams, yeast extract paste 250 grams, sal epsom 60 grams, tween 80 60 grams, potassium primary phosphate 400 grams, water 100L, be loaded in the seeding tank of 130L, 130 ℃ of sterilizings 30 minutes; After cooling, 30 ℃ of temperature, 125 rev/mins of mixing speed, air flow 1:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 50 hours;
4. wastewater degradation
The Turkey-galls of take prepares the waste liquor of gallic acid by fermentation of Aspergillus niger as raw material, through concentrated be the waste water 1000L that the content of gallic acid reaches 25 grams/L, 20 kilograms of glucose, 1.2 kilograms of soybean cake powders; Copper sulfate 10 grams, ferrous sulfate 10 grams, manganous sulfate 12 grams; Press 13%(seed liquor volume/wastewater volume) inoculation level liquid bacterial classification, 30 ℃ of temperature, 120 rev/mins of mixing speed, air flow 1:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 100 hours.Now the gallic acid clearance reaches 80.3%.
5. the preparation of thick enzyme preparation
The gallic acid waste water of aspergillus oryzae degraded, centrifugal 15 minutes of 4500rpm, obtain mycelium, and mycelium is dried, and preserves.Filtrate, hold back concentrated 70 times by the film of molecular weight 8000, and-20 ℃ of lyophilizes, obtain macromolecular activeconstituents.This activeconstituents contains following one or more enzymes: as saccharifying enzyme, proteolytic enzyme, phytase, lipase.The outer activeconstituents of thalline and born of the same parents weighs 3.5 kilograms altogether.
Embodiment 3
1. the making of test tube slant bacterial classification
Access one ring aspergillus oryzae in the potato sucrose substratum (Zhu Gejian, Wang Zhengxiang chief editor's " industrial microorganism experimental technique handbook ", 1994,367 pages) of new configuration (
aspergillus oryzae) bacterial classification (Chinese industrial microbial strains preservation management center C ICC2001), 35 ℃, incubation time 144 hours; 10 ℃ save backup.
2. liquid shaking bottle is cultivated the making of bacterial classification
Accurately take glucose 300 grams, peptone 50 grams, yeast extract paste 0 gram, sal epsom 10 grams, tween 80 100 grams, potassium primary phosphate 90 grams, water 10L, pH7.5, packing 250mL triangular flask, every bottle of 120 mL, totally 80 bottles, 140 ℃ of sterilizings 20 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification of 10 ℃ of preservations of a ring, at 35 ℃, 150 rev/mins, cultivates 72 hours;
3. the making of level liquid bacterial classification
Accurately take glucose 1500 grams, peptone 250 grams, yeast extract paste 0 gram, sal epsom 50 grams, tween 80 500 grams, potassium primary phosphate 450 grams, water 50L, be loaded in the first class seed pot of 70L, 140 ℃ of sterilizings 40 minutes; After sterilizing is cooling, 35 ℃ of temperature, 200 rev/mins of mixing speed, air flow 2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 50 hours;
4. the making of second-class liquid isolate
Accurately take glucose 15000 grams, peptone 2500 grams, yeast extract paste 0 gram, sal epsom 500 grams, tween 80 5000 grams, potassium primary phosphate 4500 grams, water 500L, be loaded in the first class seed pot of 700L, 140 ℃ of sterilizings 40 minutes; After sterilizing is cooling, 35 ℃ of temperature, 200 rev/mins of mixing speed, air flow 2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 18 hours;
5. wastewater degradation
Take Microcrystalline Cellulose, gallic acid is raw material, under the effect of catalyzer, adopts the waste liquid of direct esterification synthesizing gallic acid Microcrystalline Cellulose ester, the waste liquid 1000L that to be diluted to gallic acid content be 1 gram/L, 30 kilograms of glucose, 1.8 kilograms of soybean cake powders; Copper sulfate 15 grams, ferrous sulfate 15 grams, manganous sulfate 20 grams, press 20%(seed liquor volume/wastewater volume) inoculation level liquid bacterial classification, 34 ℃ of temperature, 190 rev/mins of mixing speed, air flow 2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 168 hours.The gallic acid clearance reaches 100%.
6. the preparation of thick enzyme preparation
The gallic acid waste water of aspergillus oryzae degraded, centrifugal 20 minutes of 6000rpm, obtain mycelium, and mycelium is dried, and preserves.Filtrate, hold back concentrated 100 times by the film of molecular weight 10000, and-20 ℃ of lyophilizes, obtain macromolecular activeconstituents.This activeconstituents contains following one or more enzymes: as saccharifying enzyme, proteolytic enzyme, phytase, lipase.The outer activeconstituents of thalline and born of the same parents weighs 2.4 kilograms altogether.
Claims (10)
1. aspergillus oryzae degraded gallic acid waster water process technology, it is characterized in that carrying out according to following step: take aspergillus oryzae as starting strain, carry out test tube enlarged culturing, liquid shaking bottle cultivation, thalline fluid enlargement culture, liquid fermenting is removed gallic acid in waste water, and the gallic acid clearance can reach 60～100%.
2. a kind of aspergillus oryzae degraded gallic acid waster water process technology according to claim 1, its feature is any strain bacterial classification of aspergillus oryzae at bacterial classification used.
3. a kind of aspergillus oryzae degraded gallic acid Technology according to claim 1, its feature is any one waste water that contains gallic acid at applicable waste water, extracts the waste liquid of gallic acid as the waste liquid of prodn. of gallic acid by fermentation process, from plant or take the waste liquid of gallic acid as synthetic other products of raw material.
4. a kind of aspergillus oryzae degraded gallic acid Technology according to claim 1, its feature enlarged culturing base in described test tube enlarged culturing therein is the potato sucrose substratum.
5. a kind of aspergillus oryzae degraded gallic acid Technology according to claim 1, its feature during described liquid shaking bottle is cultivated therein and liquid shaking bottle substratum and liquid thalline substratum in the thalline fluid enlargement culture be: glucose 5～30 grams per liters, peptone 0～5 grams per liter, yeast extract paste 0～5 grams per liter, sal epsom 0.2～1 grams per liter, tween-80 0.5～10 grams per liter, potassium primary phosphate 2～9 grams per liters, pH5～8,120～140 ℃ sterilizing 20～40 minutes.
6. a kind of aspergillus oryzae degraded gallic acid Technology according to claim 1, its feature described shake-flask culture processing condition therein is, inoculation 3-5 piece aspergillus oryzae test tube slant thalline (5 * 5mm) is in the 250mL triangular flask that 40～120 mL substratum are housed, at rotating speed: 150 rev/mins, 25～35 ℃, cultivate 24～72 hours.
7. a kind of aspergillus oryzae degraded gallic acid Technology according to claim 1, its feature described yeast culture technique therein is, by 1～20%(volume ratio) inoculum size the shake-flask culture seed liquor is inoculated in liquid thalline enlarged culturing, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 18～72 hours.
8. a kind of aspergillus oryzae degraded gallic acid waster water process technology according to claim 1, its feature in the described gallic acid wastewater medium that contains is: the waste water that contains gallic acid makes gallic acid concentration reach 1～50 grams per liter waste water 1000L through concentrated or dilution, 5～30 kilograms of glucose, 0.5～2 kilogram of soybean cake powder; Copper sulfate 5～15 grams, ferrous sulfate 5～15 grams, manganous sulfate 5～20 grams, each composition of this substratum can amplify in this ratio.
9. a kind of aspergillus oryzae degraded gallic acid waster water process technology according to claim 1, its feature described degrading waste water technique therein is: by 2～20%(seed liquor volume/wastewater volume) inoculation level liquid bacterial classification, 25～35 ℃ of temperature, 50～200 rev/mins of mixing speed, air flow 0.2～2:1 volume/volume/minute (ventilation gas volume/fermentating liquid volume/minute), cultivate 36～168 hours, i.e. the degradable gallic acid.
10. the degrading waste water obtained according to claims 1 centrifugal 5～20 minutes through 3000～6000rpm, obtain the aspergillus oryzae filament, and mycelium is dried, and preserves;
Filtrate is held back concentrated 50～100 times through molecular weight 5000～10000 films ,-20 ℃ of lyophilizes, and every liter of waste liquid can obtain 2-5 gram mycelium and bioactive molecule.
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