CN101407362B - Method for treating papermaking waste water by constructing artificial flora - Google Patents

Method for treating papermaking waste water by constructing artificial flora Download PDF

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CN101407362B
CN101407362B CN2008101586310A CN200810158631A CN101407362B CN 101407362 B CN101407362 B CN 101407362B CN 2008101586310 A CN2008101586310 A CN 2008101586310A CN 200810158631 A CN200810158631 A CN 200810158631A CN 101407362 B CN101407362 B CN 101407362B
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bacillus
cctcc
halomonas
flora
make
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CN101407362A (en
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许平
杨春玉
王照华
马翠卿
苏海军
李阳
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Shandong University
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Abstract

The invention discloses a method for processing paper-making waste water in the way of structuring artificial flora, including selecting more than two strains of Halomonas sp. flora and Bacillus sp. flora respectively for sterile culture to lead OD620nm to reach over 10.0 and then mixing the bacterial liquid by volume rate to structure the artificial flora; inoculating the paper-making waste water of pH being 9-11 and COD being 1,000-140,000mg l <-1> with the structured artificial flora, conducting stationary treatment for a certain time at 35-38 DEG C with intermittent stirring, in the treatment process, sampling at intervals to measure the pH, chroma and COD index of the paper-making waste water and finishing the treatment when the pH reduces to 7.0-8.8. The method is characterized by simple structure of the flora to be structured, obvious effect in direct treatment, strong controllability and repeatability and the like, and has great application prospect in the treatment aspects ofwaster water such as the control over the paper-making waste water.

Description

A kind of to make up the method for artificial flora Processing Paper Wastewater
Technical field
The present invention relates to utilize flora to handle the method for waste water, relate in particular to a kind of method, and obtain the operational path of two step treatment mechanisms with structure artificial flora Processing Paper Wastewater.
Background technology
Globally present rapid ascendant trend at present,,, make a large amount of forests and timber reduce gradually because pulpwood is mainly timber in developed country with the paper demand.The shortage of wood raw material resource makes papermaking straw pulp very extensive in developing country, and the papermaking straw pulp that derives from alkaline process technology has advantages such as paper quality is good, raw material is cheaply abundant.But well-known, the drawback of papermaking straw pulp maximum is to the severe contamination of environment because pollution load is high.
Be different from wood pulp waste water, straw-pulp-papermaking wastewater has the silicon content height, contains the high characteristics of saccharan, thereby it is a lot of to make the viscosity of this waste water to exceed wood pulp, therefore the method that reclaims for the suitable alkali of wood pulp waste water has characteristics such as the rate of recovery is low, cost height to handling straw-pulp-papermaking wastewater, and is especially more inapplicable for the less paper mill of the numerous scales of China.Lignin by acid separation also is a kind of method of reasonable processing waste water, but need to consume a large amount of mineral acids xylogen is precipitated, and also has the secondary pollution of inorganic acid radical ionic simultaneously.
The microbiological treatment environmental pollutant also have some reports because of it has advantages such as cost is low, secondary pollution is little on paper waste is handled.On paper waste was handled, whiterot fungi had been subjected to extensive concern owing to having stronger lignin degradation ability.But when whiterot fungi was used for wastewater treatment, wastewater treatment must just can be carried out under lower pH and COD value, and therefore a large amount of dilutions causes the practical application of this bacterium to be subjected to very big obstruction.Some bacteriums also are found has the ability of handling waste water, but also existence can not tolerate the environment of highly basic and high COD and need pretreated problem.Same, utilizing flora to come in the application of Processing Paper Wastewater, though active sludge and oxidation pond have the reasonable effect of having brought into play in the secondary treatment of paper waste, can not directly handle paper waste.Also once there were some patents to propose to adopt acid-producing bacteria group Processing Paper Wastewater, but do not provide clearly description for concrete treatment effect (as one of the most important index of waste water COD), flora characteristic and flora building process and processing mechanism.In a word, above-mentioned report and patent are not described in detail and study micro-organism treatment process condition and treatment mechanism when utilizing the flora Processing Paper Wastewater, therefore exist treating processes unclear, be difficult to shortcomings such as repetition and control, thereby make the actual development and application of these technologies have certain uncontrollability and blindness.
Summary of the invention
At above-mentioned the deficiencies in the prior art, the purpose of this invention is to provide a kind of to make up the method for artificial flora Processing Paper Wastewater.Artificial flora of the present invention not only has the ability of direct Processing Paper Wastewater, and can make the pH of waste water be reduced to neutrality by strong basicity, and colourity and COD also have certain removal.As a kind of primary treatment method, follow-up processing provides advantageous conditions to paper waste in the present invention, has strengthened organic efficiency, has reduced cost and has reduced secondary pollution.
Of the present invention to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: choose salt Zymomonas mobilis (Halomonas) the sp.Y2 CCTCC NO:M 208188 that is preserved in " Chinese typical culture collection center (CCTCC) " on October 29th, 2008, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186, salt Zymomonas mobilis (Halomonas) sp.19-DCCTCC NO:M 208193 bacterial strains and genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCCNO:M 208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187, the bacterial strain difference sterile culture of two or more in genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains is to OD 620nmReach more than 10, then in volume ratio in Halomonas sp.Y2: Bacillus sp.Y4: Bacillus sp.Y5: Bacillus sp.17-1: Bacillus sp.17-3: Bacillus sp.17-4: Halomonas sp.17-5: Halomonas sp.19-A: Bacillus sp.19-B: Halomonas sp.19-D: Bacillus sp.Y6 is that the ratio of 1-4: 1-4: 0-1: 0-1: 1-2: 0-1: 1-2: 1-2: 0-1: 0-1: 1-2 is mixed bacterium liquid, makes up artificial flora; It is 9～11 that the artificial flora that makes up is inserted pH in volume ratio according to 15%～30% inoculum size, and COD is 1,000～140,000mgl -1Paper waste in, static Processing Paper Wastewater under 35～38 ℃ of conditions, during intermittently stir, sampling and measuring paper waste pH, colourity and COD index at interval in the treating processes, when paper waste pH reduces between 7.0～8.8, processing finishes.
Further, the concrete operations step with the method that makes up the artificial flora Processing Paper Wastewater of the present invention is:
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, the bacterial strain of two or more in salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 and salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 floras carries out following operation respectively:
(1) slant culture: is 1.5～2.0% agar powders with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 15～24 hours for 30 ± 1 ℃;
(2) first order seed is cultivated: with the thalline of step (1) cultivation, aseptic condition encircles in 100ml completely liq substratum with inoculation articulating 1～4 down, under 30 ± 1 ℃ of conditions on 120～200 rev/mins of shaking tables shaking culture 15～24 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5%～8% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ± 1 ℃ of conditions on 120～200 rev/mins of shaking tables shaking culture 15～24 hours to OD 620nmReach between 10～12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3～4 times; Again be made into OD with physiological saline afterwards 620nmReach the bacteria suspension between 10～12, standby;
Simultaneously, choose genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M 208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187, the bacterial strain of two or more in genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 floras carries out following operation respectively:
(5) slant culture: is 1.5～2.0% agar powders with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 15～24 hours for 37 ± 1 ℃;
(6) first order seed is cultivated: with the thalline of step (5) cultivation, aseptic condition encircles in 100ml completely liq substratum with inoculation articulating 2 down, under 37 ± 1 ℃ of conditions on 100～150 rev/mins of shaking tables shaking culture 15～24 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5%～8% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ± 1 ℃ of conditions on 100～150 rev/mins of shaking tables shaking culture 15～24 hours to OD 620nmReach between 10～12, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3～4 times; Again be made into OD with physiological saline afterwards 620nmReach the bacteria suspension between 10～12, standby;
(9) make up artificial flora:in volume ratio; ( Halomonas ) sp.Y2 CCTCC NO：M208188∶ ( Bacillus ) sp.Y4 CCTCC NO：M 208189∶ ( Bacillus ) sp.Y5 CCTCC NO：M 208190∶ ( Bacillus ) sp.17-1 CCTCC NO：M 208183∶ ( Bacillus ) sp.17-3 CCTCC NO：M 208184∶ ( Bacillus ) sp.17-4CCTCC NO：M 208185∶ ( Halomonas ) sp.17-5 CCTCC NO：M 208192∶ ( Halomonas ) sp.19-A CCTCC NO：M 208186∶ ( Bacillus ) sp.19-B CCTCCNO：M 208187∶ ( Halomonas ) sp.19-D CCTCC NO：M 208193∶ ( Bacillus ) sp.Y6 CCTCC NO：M 2081911-4∶1-4∶0-1∶0-1∶1-2∶0-1∶1-2∶1-2∶0-1∶0-1∶1-2 ( 4 ) ( 8 ) ,；
(10) handle black liquid: the artificial flora that will make up is 9～11 in volume ratio according to 15%～30% inoculum size access pH, and COD is 1,000～140,000mgl -1Paper waste in, static Processing Paper Wastewater under 35～38 ℃ of conditions, during intermittently stir, sampling and measuring paper waste pH, colourity and COD index at interval in the treating processes, when paper waste pH reduces between 7.0～8.8, processing finishes.
Above-mentioned with in the method that makes up the artificial flora Processing Paper Wastewater: described strain culturing is used the completely liq substratum of improvement, its prescription (mass percent) is: 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH; The prescription of wherein said metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl120mmol; The prescription of described solid slant culture base is to contain the completely liq substratum that mass percent is 1.5～2.0% agar powders.
Above-mentioned substratum uses after 20 minutes in sterilization under 115 ℃ of conditions.
Above-mentioned with in the method that makes up the artificial flora Processing Paper Wastewater:the artificial flora of described structure; , ( Halomonas ) sp.Y2 CCTCC NO：M 208188∶ ( Bacillus ) sp.Y4 CCTCC NO：M 208189∶ ( Bacillus ) sp.Y5 CCTCC NO：M 208190∶ ( Bacillus ) sp.17-1 CCTCC NO：M 208183∶ ( Bacillus ) sp.17-3 CCTCCNO：M 208184∶ ( Bacillus ) sp.17-4 CCTCC NO：M 208185∶ ( Halomonas ) sp.17-5 CCTCC NO：M 208192∶ ( Halomonas ) sp.19-A CCTCC NO：M 208186∶ ( Bacillus ) sp.19-B CCTCC NO：M 208187∶ ( Halomonas ) sp.19-D CCTCC NO：M 208193∶ ( Bacillus ) sp.Y6 CCTCC NO：M 2081914∶2∶1∶1∶2∶1∶1∶2∶1∶1∶2 ( 4 ) ( 8 ) 。
If use the artificial flora of incomplete strain construction of the present invention; Preferably in volume ratio, in Halomonas (Halomonas) sp.Y2 CCTCC NO:M 208188: bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189: bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184: Halomonas (Halomonas) sp.17-5 CCTCC NO:M 208192: Halomonas (Halomonas) sp.19-A CCTCC NO:M 208186: bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 is 2: 1: 1: 1: 1: 1 ratio is mixed the bacterium liquid that step (4) and step (8) make.
Above-mentioned in the method that makes up the artificial flora Processing Paper Wastewater: the artificial flora of the described structure of step (10) preferably inserts in the paper waste static Processing Paper Wastewater under 37 ℃ of conditions with volume ratio according to 20% inoculum size.
Wherein: preferred 15～30 minutes of the churning time of the described stirring at intermittence of step (10), rotating speed is 50～120 rev/mins; Intermittent time is 4～8 hours.
The result of measuring pH, colourity and COD index shows:
Artificial flora of the present invention has obvious reduction pH, removes the ability of part colourity and COD paper waste water; The flora of analyzing in the wastewater treatment process in conjunction with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) changes, and confirms that also flora of the present invention is one group of relatively stable flora with direct Processing Paper Wastewater ability; Paper waste before and after handling is used ethyl acetate and chloroform extraction respectively three times, it is heavy molten after the method that adopts nitrogen to blow is all volatilized organic solvent with the 1ml solvent, the compound component that GC-MS (gas chromatography mass spectrometry) analyzes the processed front and back of paper waste changes, monomeric compound such as methyl catechol, the vanillin food grade,1000.000000ine mesh etc. of compound that some of them are natural such as cadinol, tetradecenoic acid and xylogen are detected, these compounds all have reduction significantly after being handled by flora, illustrate that artificial flora of the present invention has the ability of these compounds of degraded.
Paper waste is that a kind of microorganism is difficult to the directly waste water of processing, the waste water after handling through the complete flora of the present invention's structure, nearly 7.5gl -1Formic acid, 8.9gl -1Acetate and 2.5gl -1Lactic acid produces, and pH can be reduced to below 8.0 from 11.0; Colourity and the 18.5%COD of while about 35% CrChroma in waste water be removed, waste water is become brown by black.Its viscosity of waste water after the processing obviously reduces, and viscosity is reduced to about 4.3CP after the processing by the 8.68CP before handling after testing.
Further utilize degradation curve and PCR-DGGE molecular ecology analytical procedure to detect the dynamic change that flora is handled each bacterial strain in the waste water process, show that treating processes of the present invention is the mechanism of two step processing modes: promptly initial stage Halomonassp. plays a significant role on product acid reduction waste water ph, has certain decolorization simultaneously; Later stage Bacillus sp. has brought into play vital role in some lignin monomer degradation, colourity further is removed when COD reduces, and reason may be that some chromophoric grouies that contain in the xylogen are degraded.
Unusual effect of the present invention and advantage are:
(1) flora of Gou Jianing has the significant ability of directly handling high pH, high COD paper waste, and waste water does not need dilution, makes this flora have great potential in application;
(2) adopt aerobic and array mode facultative anaerobe makes up artificial flora, taken into account aerobic and facultative anaerobe to the not same-action of paper waste, improved biodegradability, avoided the limitation of single bacterial strain processing power;
(3) be different from domestication floras such as active sludge and oxidation pond, the artificial flora that the present invention makes up is simple in structure, control easily, and good reproducibility, and select for use bacterial strain to be easy to cultivate.
(4) the two step Processing Paper Wastewater mechanism that experimental results show that through PCR-DGGE will improve the application potential that makes up flora to the practical application performance important directive function of described flora in wastewater treatment.
In a word, this structure flora has very big application potential in the direct primary treatment of paper waste, provides favourable condition to the post-processed of waste water.
Description of drawings
Halomonas sp.Y2 provided by the invention, Halomonas sp.17-5, Halomonas sp.19-A, Halomonas sp.19-D bacterial strain and Bacillus sp.Y4, Bacillus sp.Y5, Bacillus sp.17-1, Bacillus sp.17-3, Bacillus sp.17-4, Bacillus sp.19-B, Bacillus sp.Y6 bacterial strain have been preserved in " Chinese typical culture collection center (CCTCC) ", preservation address on October 29th, 2008: Wuhan City Wuhan University China typical culture collection center, postcode: 430072, its deposit number is: CCTCC NO:M 208183～CCTCC NO:M 208193.
Fig. 1 microflora degradation pH 11.0, COD 140,000mg l -1The degradation curve of original waste water.
Wherein: A, the pH value of handling waste water changes (zero); The colourity of pH7.6 contrast changes (); The colourity of handling waste water changes (■); Handle the COD of waste water Cr(△); B, the lactic acid concn (▲) in the wastewater treatment process; Formic acid concn in the wastewater treatment process (◇); Acetic acid concentration (◆) in the wastewater treatment process.
Fig. 2 PCR-DGGE collection of illustrative plates detects the flora dynamic change in the wastewater treatment process.
Wherein: swimming lane 1, flora structure collection of illustrative plates before handling; Swimming lane was handled the back collection of illustrative plates in 2,44 hours; Swimming lane was handled the back collection of illustrative plates in 3,68 hours; Swimming lane was handled the back collection of illustrative plates in 4,92 hours; Swimming lane was handled the back collection of illustrative plates in 5,116 hours; Swimming lane was handled the back collection of illustrative plates in 6,140 hours; Swimming lane was handled the back collection of illustrative plates in 7,180 hours.
Fig. 3 uses GC-MS to detect the composition variation that flora is handled waste water front and back waste water.
Wherein: sample adopts ethyl acetate extraction to handle the back and concentrates, and uses the pillar of DB-1 to detect.
Fig. 4 uses GC-MS to detect the composition variation that the flora two-step approach is handled waste water front and back waste water.
Wherein: sample adopts chloroform extraction to handle the back and concentrates, and uses the pillar of DB-5 to detect.A: contrast before handling; B: flora was handled 5 days.
Embodiment
Embodiment 1: each strain growth is to the mensuration of demand, tolerable temperature, pH and the tolerance of salinity of oxygen
(1) bacterial classification is selected: select for use from environment separation such as various extreme physical environments such as the high salt of highly basic to and be preserved in " four strain salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCCNO:M 208188 of Chinese typical culture collection " center "; salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192; salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 or salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 and seven bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189; genus bacillus (Bacillus) sp.Y5CCTCC NO:M 208190; genus bacillus (Bacillus) sp.17-1 CCTCC NO:M 208183; genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184 on October 29th, 2008, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187 or genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191;
Choose above-mentioned Halomonas sp.Y2 at first respectively, Halomonas sp.17-5, Halomonas sp.19-A, the bacterial strain in the Halomonas sp.19-D flora carries out following operation respectively:
(2) Halomonas slant culture: is that 1.6% agar powder, pH are on 8.0 the LB substratum with above-mentioned Halomonas inoculation in containing mass percent, cultivates thalline 20 hours for 30 ℃;
(3) Halomonas seed culture: with the thalline that step (2) is cultivated, aseptic condition encircles in the triangular flask of the 500ml that contains 100ml LB liquid nutrient medium with inoculating articulating 2 down, under 30 ℃ of conditions on shaking table 200 rev/mins of shaking culture 16 hours, make seed.With centrifugal 8 minutes of thalline 5000rpm, collect the thalline that each bacterium obtains respectively, suspend with physiological saline with after the physiological saline washing 3 times;
(4) in definite durothermic experiment of each Halomonas bacterial strain, the experiment substratum uses the LB substratum, and filling a prescription is (mass percent): 1% peptone, 0.5% yeast extract paste, 1% NaCl, adding distil water is to 1L, pH transfers under 9.5,115 ℃ of conditions with 4M NaOH and sterilized 20 minutes.Inoculum size 5%, growth temperature are respectively 4 ℃, 10 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, 45 ℃, 50 ℃, 55 ℃, and the 200rpm shaking culture is 24 hours on shaking table, measure OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The temperature tolerance result of each bacterial strain as shown in Table 1.The temperature growth scope of Halomonas is 4-45 ℃, and optimum growth temp is 30 ℃ or 37 ℃.
(5) in definite alkali-proof experiment of each Halomonas bacterial strain, the experiment substratum uses the LB substratum, prescription is (mass percent): 1% peptone, 0.5% yeast extract paste, 1% NaCl adds pH and is respectively 5.0,6.0,7.0,8.0,9.0,9.5,10.0,10.5,11.0 and 12.0 KH 2PO 4/ K 2HPO 4OrNa 2CO 3/ NaHCO 3Damping fluid 1L preparation.Sterilization is 20 minutes under 115 ℃ of conditions.5%, 30 ℃ of inoculum size 200rpm shaking culture 24 hours on shaking table is measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The alkali resistance result of each bacterial strain as shown in Table 1, experimental strain is the alkali resistance bacterial strain, and can optimum growh at pH about 10.
(6) in the experiment of determining each Halomonas bacterial strain tolerance of salinity, the experiment substratum uses the LB substratum, prescription is (mass percent): 1% peptone, 0.5% yeast extract paste, add mass percent respectively and be 0%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 18%, 20% NaCl, pH transfers under 9.5,115 ℃ of conditions with 4M NaOH and sterilized 20 minutes.5%, 30 ℃ of inoculum size 200rpm shaking culture 24 hours on shaking table is measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.Experimental result as shown in Table 1, the result shows that Halomonas sp. is a moderate salt tolerant bacterium.Y2 wherein, 17-5 and 19-A can grow under the NaCl of 0-18% concentration.
(7) in the experiment of determining each Halomonas bacterial strain aerobism, the experiment substratum uses the LB substratum, and filling a prescription is (mass percent): 1% peptone, and 0.5% yeast extract paste, 1% NaCl, pH are adjusted to 8.0 or 10.0 respectively.Sterilization is 20 minutes under 115 ℃ of conditions, inoculum size 5% (v/v), and 30 ℃ of 200rpm shaking culture 24 hours on shaking table are measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The result shows that Halomonas is aerobic bacterial strain.
Choose above-mentioned Bacillus sp.Y4 again, Bacillus sp.Y5, Bacillus sp.17-1, Bacillus sp.17-3, Bacillus sp.17-4, Bacillus sp.19-B, the bacterial strain in the Bacillus sp.Y6 flora carries out following operation respectively:
(8) Bacillus bacterial strain slant culture: is that 1.6% agar powder, pH are on 8.0 the LB substratum with above-mentioned Bacillus inoculation in containing mass percent, 37 ℃, 100rpm shaking culture thalline 20 hours;
(9) Bacillus seed culture: with the thalline that step (8) is cultivated, aseptic condition encircles in the triangular flask of the 500ml that contains 100ml LB liquid nutrient medium with inoculating articulating 2 down, under 37 ℃ of conditions on shaking table 100 rev/mins of shaking culture 24 hours, make seed.With centrifugal 8 minutes of thalline, collect the thalline that each bacterium obtains respectively, suspend with physiological saline with after the physiological saline washing 3 times;
(10) in definite durothermic experiment of each Bacillus bacterial strain, the experiment substratum uses the LB substratum, and filling a prescription is (mass percent): 1% peptone, 0.5% yeast extract paste, 1% NaCl, adding distil water is to 1L, pH transfers under 8.0,115 ℃ of conditions with 4M NaOH and sterilized 20 minutes.Inoculum size 5%, growth temperature are respectively 4 ℃, 10 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, 45 ℃, 55 ℃, 60 ℃ and 65 ℃, and the 100rpm shaking culture is 24 hours on shaking table, measure OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The temperature tolerance result of each bacterial strain as shown in Table 1.The Bacillus growth temperature range is 10-60 ℃, and optimum growth temp is 30 ℃ or 42 ℃.
(11) in definite alkali-proof experiment of each Bacillus bacterial strain, it is (mass percent) that the experiment substratum uses the LB culture medium prescription: 1% peptone, 0.5% yeast extract paste, 1% NaCl adds pH and is respectively 5.0,6.0,7.0,8.0,9.0,9.5,10.0,10.5,11.0 and 12.0 KH 2PO 4/ K 2HPO 4Or Na 2CO 3/ NaHCO 3Damping fluid 1L preparation.Sterilization is 20 minutes under 115 ℃ of conditions.5%, 37 ℃ of inoculum size 100rpm shaking culture 24 hours on shaking table is measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The alkali resistance result of each bacterial strain as shown in Table 1, experimental strain is the alkali resistance bacterial strain, and can optimum growh at pH8-9.
(12) in the experiment of determining each Bacillus bacterial strain tolerance of salinity, the experiment substratum uses the LB substratum, prescription is (mass percent): 1% peptone, 0.5% yeast extract paste, add mass percent respectively and be 0%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 18%, 20% NaCl, pH transfers under 9.5,115 ℃ of conditions with 4M NaOH and sterilized 20 minutes.5%, 37 ℃ of inoculum size 100rpm shaking culture 24 hours on shaking table is measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.Experimental result as shown in Table 1, the result shows that the big multipotency of Bacillus obtains optimum growh under the NaCl of 0-1%.
(13) in the experiment of determining each Bacillus bacterial strain aerobism, the experiment substratum uses the LB substratum, and filling a prescription is (mass percent): 1% peptone, and 0.5% yeast extract paste, 1% NaCl, pH are adjusted to 8.0 or 10.0 respectively.Sterilization is 20 minutes under 115 ℃ of conditions, and 5%, 37 ℃ of inoculum size 100rpm shaking culture 24 hours on shaking table is measured OD 600nmValue.OD 600nmBe considered as the positive greater than 0.3.The result shows that Bacillus is the amphimicrobian bacterial strain.
Table one is selected the growth conditions of bacterial strain
Figure G2008101586310D00071
Embodiment 2: make up artificial flora and carry out the method that paper waste is handled
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188 and salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours to OD 620nmReach 10, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
Simultaneously, choose genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189 and genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 16 hours to OD 620nmReach 10, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, according to Halomonas sp.Y2: Bacillus sp.Y4: Bacillus sp.17-3: Halomonas sp.19-A is 1: 1: the ratio of 1:1 is mixed the bacterium liquid that step (4) and step (8) make, and makes up artificial flora;
(10) handle black liquid: the artificial flora that will make up is 9 in volume ratio according to 15% inoculum size access pH, and COD is 1,000mgl -1Paper waste in, static Processing Paper Wastewater under 35 ℃ of conditions, during intermittently stir that (churning time is 15 minutes, and rotating speed is 50 rev/mins; Intermittent time is 4 hours), at interval sampling and measuring paper waste pH, colourity and COD index in the treating processes, when paper waste pH reduces to 7.0 and stablize when constant, processing finishes.
PH adopts the Ao Lilong acidometer to detect; Organic acid adopts HPLC, and (Agilent 1100 series Hewlett-Packard) detect; Colourity adopts 2100 spectrophotometers to detect the absorbance value of OD under 465nm; COD adopts potassium dichromate process to detect.Experimental result shows: pH drops to about 7.0 by 9.0.About 38% colourity and 70.5%COD are removed.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl 120 mmol.
Embodiment 3: make up flora and carry out the method that paper waste is handled
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192 and salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 15 hours to OD 620nmReach 11, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmBe 11 bacteria suspension, standby;
Simultaneously, choose genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184 and genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours to OD 620nmReach 10, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmBe 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, according to Halomonas sp.Y2: Bacillus sp.Y4: Bacillus sp.17-3: Halomonas sp.17-5: Halomonas sp.19-A: Bacillus sp.Y6 is 2: 1: 1: 1: 1: 1 ratio is mixed the bacterium liquid that step (4) and step (8) make, and makes up artificial flora;
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 30% inoculum size access pH be 11, COD is 100,000mg1 -1Paper waste in, static Processing Paper Wastewater under 37 ℃ of conditions, during intermittently stir that (churning time is 25 minutes, and rotating speed is 120 rev/mins; Intermittent time is 6 hours), at interval sampling and measuring paper waste pH, colourity and COD index in the treating processes, when paper waste pH reduces to 8.4 and stablize when constant, processing finishes.
PH adopts the Ao Lilong acidometer to detect; Organic acid adopts HPLC, and (Agilent 1100 series Hewlett-Packard) detect; Colourity adopts 2100 spectrophotometers to detect the absorbance value of OD under 465nm; COD adopts potassium dichromate process to detect.Experimental result shows: pH reduces to about 8.4 by 11.0.About 29% colourity and 10.8% COD are removed.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl120mmol.
Embodiment 4: make up flora and carry out the method that paper waste is handled
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188 and salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours to OD 620nmReach 12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 12 bacteria suspension, standby;
Simultaneously, choose genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M 208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184 and genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 20 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 20 hours to OD 620nmReach 10, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, according to the ratio of Halomonas sp.Y2:Bacillus sp.Y4:Bacillussp.17-1:Bacillus sp.17-4:Bacillus sp.17-3:Halomonas sp.19-A=1:1:1:1:1:1 the bacterium liquid that step (4) and step (8) make is mixed, make up artificial flora;
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 30% inoculum size access pH be 11, COD is 100,000mg l -1Paper waste in, static Processing Paper Wastewater under 37 ℃ of conditions, during intermittently stir that (churning time is 15 minutes, and rotating speed is 120 rev/mins; Intermittent time is 8 hours), at interval sampling and measuring paper waste pH, colourity and COD index in the treating processes, when paper waste pH reduces to 8.8 and stablize when constant, processing finishes.
PH adopts the Ao Lilong acidometer to detect; Organic acid adopts HPLC, and (Agilent 1100 series Hewlett-Packard) detect; Colourity adopts 2100 spectrophotometers to detect the absorbance value of OD under 465nm; COD adopts potassium dichromate process to detect.Experimental result shows: pH reduces to about 8.8 by 11.0.About 28% colourity and 10.5% COD are removed.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl 120 mmol.
Embodiment 5: make up flora and carry out the method that paper waste is handled
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 and salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 15 hours to OD 620nmReach 12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 12 bacteria suspension, standby;
Simultaneously, choose seven bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187 and genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours to OD 620nmReach 10, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, (the bacterium liquid that step (4) and step (8) are made mixes, and makes up artificial flora according to the ratio of Halomonas sp.Y2:Bacillus sp.Y4:Bacillussp.Y5:Bacillus sp.17-1:Bacillus sp.17-3:Bacillus sp.17-4:Halomonas sp.17-5:Halomonas sp.19-A:Bacillus sp.19-B:Halomonas sp.19-D:Bacillus sp.Y6=4:2:1:1:2:1:1:2:1:1:2;
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 20% inoculum size access pH be 11, COD is 140,000mg l -1Paper waste in, static Processing Paper Wastewater and intermittently stir that (churning time is 30 minutes, and rotating speed is 60 rev/mins under 37 ℃ of conditions; Intermittent time is 4 hours) sampling and measuring paper waste pH, colourity and COD index at interval in the treating processes, when paper waste pH reduces to 7.8 and stablize when constant, processing finishes.Every four hours interval sampling and measuring pH, organic acid, colourity and COD indexs.Result shown in Figure 1 shows that this flora has direct Processing Paper Wastewater and produces the ability that acid reduces pH, removes part colourity and COD.PH adopts the Ao Lilong acidometer to detect; Organic acid adopts HPLC, and (Agilent1100series Hewlett-Packard) detects; Colourity adopts 2100 spectrophotometers to detect the absorbance value of OD under 465nm; COD adopts potassium dichromate process to detect.
Can find (Fig. 1) by the change curve of observing the pH value, the entire treatment process has been divided into two stages: the fs, organic acid mainly was created in this stage treatment stage of being 0-68h, approximately 3.5gl -1Lactic acid, 6.7gl -1Formic acid and 6.5gl -1Acetate produces, and makes pH be reduced to 7.8 by 11.0.In this stage, reach 18% colourity and 16 approximately, 000mgl -1COD Cr(11.5%) is removed; The treatment stage that subordinate phase being 68-180, increased 2.2gl at this stage processing acetate -1In addition, formic acid and lactic acid concn do not have to change the colourity and 10 in this stage nearly 17.5%, 000mgl substantially -1COD Cr(7.0%) further removed.In general, about 35% colourity and 18.5%COD CrBe removed in the entire treatment process.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl 120 mmol.
Embodiment 6:PCR-DGGE detects the dynamic change of handling flora in the waste water process
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 and salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 15 hours to OD 620nmReach 12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 12 bacteria suspension, standby;
Simultaneously, choose seven bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187 and genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours to OD 620nM reaches 10, makes secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, (the bacterium liquid that step (4) and step (8) are made mixes, and makes up artificial flora according to the ratio of Halomonas sp.Y2:Bacillus sp.Y4:Bacillussp.Y5:Bacillus sp.17-1:Bacillus sp.17-3:Bacillus sp.17-4:Halomonas sp.17-5:Halomonas sp.19-A:Bacillus sp.19-B:Halomonas sp.19-D:Bacillus sp.Y6=4:2:1:1:2:1:1:2:1:1:2;
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 20% inoculum size access pH be 11, COD is 140,000mgl -1Paper waste in, static Processing Paper Wastewater under 37 ℃ of conditions, during intermittently stir that (churning time is 30 minutes, and rotating speed is 60 rev/mins; Intermittent time is 4 hours);
(11) in conjunction with polymerase chain reaction-denaturing gradient gelelectrophoresis (PCR-DGGE, Bio-Rad, Hercules, CA) flora of analyzing in the wastewater treatment process changes, thereby determines that this flora is a relatively stable flora with direct Processing Paper Wastewater ability.
PCR-DGGE collection of illustrative plates (Fig. 2) shows that in the waste water batch operation Halomonas bacterial strain has the ability of dominant growth when handling early stage, but relatively stable in the later stage.The Bacillus bacterial strain has stronger growth process ability early stage in the inactive later stage, and the conclusion that the degradation curve among this and the embodiment 4 draws meets.
In view of the above, this makes up flora and exists one two step to handle mechanism when Processing Paper Wastewater: handle early stage when conditions such as oxygen, saccharan are more competent, four strain Halomonas support under the condition the eugonic bacterial strain growth conditions of having the advantage as a class well in strictness, produce acid and make the pH of waste water be reduced to slight alkalinity by consuming various glucides; Growth for amphimicrobian Bacillus bacterium when pH is reduced to a certain degree provides more satisfactory condition, and seven strain Bacillus bacterium begin to play a role in wastewater treatment, consumes some waste water components than difficult degradation COD and colourity are further reduced.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl 120 mmol.
Embodiment 7:GC-MS detects the variation that flora is handled the front and back waste water composition
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 and salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 bacterial strains carry out following operation respectively:
(1) slant culture: is 1.6% agar powder with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 20 hours for 30 ℃;
(2) first order seed is cultivated: with the thalline that step (1) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 16 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ℃ of conditions on 200 rev/mins of shaking tables shaking culture 15 hours to OD 620nmReach 12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 12 bacteria suspension, standby;
Simultaneously, choose seven bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187 and genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains carry out following operation respectively:
(5) slant culture: is 1.6% agar powder with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 24 hours for 37 ℃;
(6) first order seed is cultivated: with the thalline that step (5) is cultivated, aseptic condition encircles in 100ml completely liq substratum with inoculating articulating 2 down, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ℃ of conditions on 100 rev/mins of shaking tables shaking culture 18 hours to OD 620nmReach 10, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3 times; Again be made into OD with physiological saline afterwards 620nmReach 10 bacteria suspension, standby;
(9) make up artificial flora: in volume ratio, (the bacterium liquid that step (4) and step (8) are made mixes, and makes up artificial flora according to the ratio of Halomonas sp.Y2:Bacillus sp.Y4:Bacillussp.Y5:Bacillus sp.17-1:Bacillus sp.17-3:Bacillus sp.17-4:Halomonas sp.17-5:Halomonas sp.19-A:Bacillus sp.19-B:Halomonas sp.19-D:Bacillus sp.Y6=4:2:1:1:2:1:1:2:1:1:2;
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 20% inoculum size access pH be 11, COD is 140,000mgl -1Paper waste in, static Processing Paper Wastewater under 37 ℃ of conditions, during intermittently stir that (churning time is 30 minutes, and rotating speed is 60 rev/mins; Intermittent time is 4 hours);
(11) sample separation is handled: the 100ml waste water before and after will handling is used ethyl acetate and chloroform extraction respectively three times, and the method that adopts nitrogen to blow makes the organic solvent back of all volatilizing heavy molten with the 1ml solvent, is used for the GC-MS analysis.
(12) GC-MS (gas chromatography mass spectrometry) detects: the compound component that GC-MS analyzes the processed front and back of waste water changes.The waste water that ethyl acetate is handled reaches the contrast waste water that does not add bacterium, and (the DB-1 chromatographic column ShimadzuGCMS-QP2010) detects, and waste water component collection of illustrative plates as shown in Figure 3 before and after handling with GC-MS; The waste water that chloroform is handled reaches the contrast waste water that does not add bacterium and detects with GC-MS (DB-5 chromatographic column, VARIAN Saturn2000).Waste water change of component before and after handling as shown in Figure 4.Fig. 3 shows and contains a lot of natural compoundss such as cadinol etc. in the waste water and a lot of natural compounds can both be by microflora degradation.Fig. 4 shows that the monomeric compound of some xylogen can analyze out by chloroform extraction, GC-MS, and some compounds such as vanillin food grade,1000.000000ine mesh etc. almost can be degraded fully.
Make up in the method for flora Processing Paper Wastewater in above-mentioned utilization, strain culturing is used the perfect medium of improvement, prescription is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, 1.6% agar powder, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.Liquid seeds is cultivated the completely liq substratum with improvement, and filling a prescription to mass percent is 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water are to 1L, and pH transfers to 8.0 with 4M NaOH.Sterilization is 20 minutes under 115 ℃ of conditions.The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O0.5g; Na 2MoO 42H 2O0.1g; CuCl 22H 2O0.05g; Na 2WO 42H 2O0.05g; HCl 120 mmol.

Claims (7)

1. one kind to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: choose and be preserved in " salt Zymomonas mobilis (Halomonas) the sp.Y2 CCTCC NO:M 208188 of Chinese typical culture collection " center "; salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192; salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186; salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 bacterial strains and genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189; genus bacillus (Bacillus) sp.Y5CCTCC NO:M 208190; genus bacillus (Bacillus) sp.17-1 CCTCC NO:M 208183 on October 29th, 2008, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4 CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187, the bacterial strain difference sterile culture of two or more in genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 bacterial strains is to OD 620nmReach more than 10.0, then in volume ratio in Halomonas sp.Y2: Bacillus sp.Y4: Bacillus sp.Y5: Bacillus sp.17-1: Bacillus sp.17-3: Bacillus sp.17-4: Halomonas sp.17-5: Halomonas sp.19-A: Bacillus sp.19-B: Halomonas sp.19-D: Bacillus sp.Y6 is that the ratio of 1-4: 1-4: 0-1: 0-1: 1-2: 0-1: 1-2: 1-2: 0-1: 0-1: 1-2 is mixed bacterium liquid, makes up artificial flora; The artificial flora that makes up is inserted pH=9-11, COD1,000-140,000mg l in volume ratio according to 15%～30% inoculum size -1Paper waste in, static processing straw-pulp-papermaking wastewater under 35～38 ℃ of conditions, during intermittently stir, sampling and measuring paper waste pH, colourity and COD index at interval in the treating processes, when paper waste pH reduces between 7.0～8.8, processing finishes.
2. according to claim 1 to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: the concrete operations step of described method is:
Choose salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188, salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192, the bacterial strain of two or more in salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186 and salt Zymomonas mobilis (Halomonas) sp.19-D CCTCC NO:M 208193 floras carries out following operation respectively:
(1) slant culture: is 1.5～2.0% agar powders with above-mentioned Halomonas inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 15～24 hours for 30 ± 1 ℃;
(2) first order seed is cultivated: with the thalline of step (1) cultivation, aseptic condition encircles in 100ml completely liq substratum with inoculation articulating 1～4 down, under 30 ± 1 ℃ of conditions on 120～200 rev/mins of shaking tables shaking culture 15～24 hours make first order seed to logarithmic phase mid-term;
(3) enlarged culturing: with volume ratio is 5%～8% inoculum size, connects first order seed in 1000ml completely liq substratum, under 30 ± 1 ℃ of conditions on 120～200 rev/mins of shaking tables shaking culture 15～24 hours to OD 620nmReach between 10～12, make secondary seed;
(4) collect the Halomonas thalline: under the 5000rpm condition centrifugal 5～8 minutes, collect the thalline that step (3) makes respectively, and with physiological saline washing 3～4 times; Again be made into OD with physiological saline afterwards 620nmReach the bacteria suspension between 10～12, standby;
Simultaneously, choose genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189, genus bacillus (Bacillus) sp.Y5 CCTCC NO:M 208190, genus bacillus (Bacillus) sp.17-1 CCTCC NO:M 208183, genus bacillus (Bacillus) sp.17-3 CCTCC NO:M 208184, genus bacillus (Bacillus) sp.17-4CCTCC NO:M 208185, genus bacillus (Bacillus) sp.19-B CCTCC NO:M 208187, the bacterial strain of two or more in genus bacillus (Bacillus) sp.Y6 CCTCC NO:M 208191 floras carries out following operation respectively:
(5) slant culture: is 1.5～2.0% agar powders with above-mentioned Bacillus inoculation in containing mass percent, and pH is on 8.0 the solid slant culture base, cultivates thalline 15～24 hours for 37 ± 1 ℃;
(6) first order seed is cultivated: with the thalline of step (5) cultivation, aseptic condition encircles in 100ml completely liq substratum with inoculation articulating 1～4 down, under 37 ± 1 ℃ of conditions on 100～150 rev/mins of shaking tables shaking culture 15～24 hours make first order seed to logarithmic phase mid-term;
(7) enlarged culturing: with volume ratio is 5%～8% inoculum size, connects first order seed in 1000ml completely liq substratum, under 37 ± 1 ℃ of conditions on 100～150 rev/mins of shaking tables shaking culture 15～24 hours to OD 620nmReach between 10～12, make secondary seed;
(8) collect the Bacillus thalline: under the 5000rpm condition centrifugal 5～8 minutes, collect the thalline that step (7) makes respectively, and with physiological saline washing 3～4 times; Again be made into OD with physiological saline afterwards 620nmReach the bacteria suspension between 10～12, standby;
(9) make up artificial flora:in volume ratio; ( Halomonas ) sp.Y2 CCTCC NO：M208188∶ ( Bacillus ) sp.Y4 CCTCC NO：M 208189∶ ( Bacillus ) sp.Y5 CCTCC NO：M 208190∶ ( Bacillus ) sp.17-1 CCTCC NO：M 208183∶ ( Bacillus ) sp.17-3 CCTCC NO：M 208184∶ ( Bacillus ) sp.17-4CCTCC NO：M 208185∶ ( Halomonas ) sp.17-5 CCTCC NO：M 208192∶ ( Halomonas ) sp.19-A CCTCC NO：M 208186∶ ( Bacillus ) sp.19-B CCTCCNO：M 208187∶ ( Halomonas ) sp.19-D CCTCC NO:M 208193∶ ( Bacillus ) sp.Y6 CCTCC NO：M 2081911-4∶1-4∶0-1∶0-1∶1-2∶0-1∶1-2∶1-2∶0-1∶0-1∶1-2 ( 4 ) ( 8 ) ,；
(10) handle black liquid: the artificial flora that will make up in volume ratio according to 15%～30% inoculum size access pH=9～11, COD1,000～140,000mg l -1Straw-pulp-papermaking wastewater in, static processing straw-pulp-papermaking wastewater under 35～38 ℃ of conditions, during intermittently stir, sampling and measuring paper waste pH, colourity and COD index at interval in the treating processes, when paper waste pH reduces between 7.0～8.8, processing finishes.
As described in the claim 2 to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: the prescription of described completely liq substratum is (mass percent): 1% glucose, 1% peptone, 0.5% yeast extract paste, 1% extractum carnis, the NaCl of 1-3%, the metal ion mixed solution of 1ml, adding distil water is to 1L, and pH transfers to 8.0 with 4M NaOH; The prescription of wherein said metal ion mixed solution is that every liter of distilled water contains: ZnCl 20.5g; FeCl 20.5g; MnCl 24H 2O 0.5g; Na 2MoO 42H 2O 0.1g; CuCl 22H 2O 0.05g; Na 2WO 42H 2O 0.05g; HCl120mmol; The prescription of described solid slant culture base is to contain the completely liq substratum that mass percent is 1.5～2.0% agar powders.
4. as claimed in claim 2 to make up the method for artificial flora Processing Paper Wastewater; It is characterized in that:the artificial flora of described structure; , ( Halomonas ) sp.Y2 CCTCC NO：M 208188∶ ( Bacillus ) sp.Y4 CCTCC NO：M 208189∶ ( Bacillus ) sp.Y5 CCTCCNO：M 208190∶ ( Bacillus ) sp.17-1 CCTCC NO：M 208183∶ ( Bacillus ) sp.17-3 CCTCC NO：M 208184∶ ( Bacillus ) sp.17-4 CCTCC NO：M 208185∶ ( Halomonas ) sp.17-5 CCTCC NO：M 208192∶ ( Halomonas ) sp.19-ACCTCC NO：M 208186∶ ( Bacillus ) sp.19-B CCTCC NO：M 208187∶ ( Halomonas ) sp.19-D CCTCC NO：M 208193∶ ( Bacillus ) sp.Y6 CCTCC NO：M 2081914∶2∶1∶1∶2∶1∶1∶2∶1∶1∶2 ( 4 ) ( 8 ) 。
As described in the claim 2 to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: the artificial flora of described structure, in volume ratio, in salt Zymomonas mobilis (Halomonas) sp.Y2 CCTCC NO:M 208188: genus bacillus (Bacillus) sp.Y4 CCTCC NO:M 208189: genus bacillus (Bacillus) sp.17-3CCTCC NO:M 208184: salt Zymomonas mobilis (Halomonas) sp.17-5 CCTCC NO:M 208192: salt Zymomonas mobilis (Halomonas) sp.19-A CCTCC NO:M 208186: genus bacillus (Bacillus) sp.Y6 CCTCCNO:M 208191 is 2: 1: 1: 1: 1: 1 ratio is mixed the bacterium liquid that step (4) and step (8) make.
As described in the claim 2 to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: the artificial flora of the described structure of step (10) inserts in the straw-pulp-papermaking wastewater according to 20% inoculum size in volume ratio, static processing straw-pulp-papermaking wastewater under 37 ℃ of conditions.
As described in the claim 2 to make up the method for artificial flora Processing Paper Wastewater, it is characterized in that: the churning time that step (10) stirs described intermittence is 15～30 minutes, and rotating speed is 50～120 rev/mins; Intermittent time is 4～8 hours.
CN2008101586310A 2008-11-04 2008-11-04 Method for treating papermaking waste water by constructing artificial flora Expired - Fee Related CN101407362B (en)

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