CN103627657B - A kind of composite flora for lignin degrading waste water and preparation method thereof - Google Patents
A kind of composite flora for lignin degrading waste water and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of composite flora for lignin degrading waste water and preparation method thereof.Does the method first distinguish picking edaphic bacillus (Agrobacterium? sp.), rod bacterium (Bacillus? sp.), enterobacter cloacae (Enterobacter.cloacae.), Gordonia bronchialis (Gordonia? sp.), pseudomonas putida (Pseudomonas? putida.), Pseudomonas stutzeri (Pseudomonas? stutzeri.) inoculation culture; By volume percentages, gets 3 ~ 6% edaphic bacilluss respectively, 3 ~ 7% rod bacteriums, 10 ~ 26% enterobacter cloacaes, 34 ~ 45% Gordonia bronchialis, and after 16 ~ 38% pseudomonas putidas and 12 ~ 23% Pseudomonas stutzeris activate, mixing is put in waste water.The present invention, by the coordinative role between bacterial classification, effectively can improve the degradation efficiency of microbiological treatment xylogen.
Description
Technical field
The present invention relates to a kind of xylogen wastewater treatment, be specifically related to a kind of composite flora for lignin degrading waste water and preparation method thereof.
Background technology
Biological treatment is current conventional method of wastewater treatment, and the pollution substance in waste water, by the metabolism of microorganism, decomposes, absorbs, thus reach the object of pollution administration by this method.Biological treatment is compared with additive method, and its cost is low, and efficiency is high, and easily operates, and the most important thing is do not have secondary pollution, therefore, is widely used in the treatment of waste water.Along with expanding economy, the composition of waste water is day by day complicated, especially when organic pollutant containing poisonous, difficult degradation in waste water, owing to having microorganism kind in the environment, the comparatively small amt of special degradation capability to this type organic, it is in a disadvantageous position in interspecific competition simultaneously, therefore, traditional biologic treating technique faces big challenge.
Due to the difficult for biological degradation of xylogen, the biochemical treatment efficiency of paper-making effluent is lower, along with paper-making effluent emission standard carry mark, effectively can remove COD, BOD in paper-making effluent and toxic substance becomes most important, the degraded how effectively improving BOD and COD, reduce follow-up materialization system chemical add and working cost becomes the technical problem that prior art is badly in need of solving.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of degradation efficiency of xylogen high, composite flora for lignin degrading waste water that expense is low and preparation method thereof.
Object of the present invention is achieved through the following technical solutions:
For a preparation method for the composite flora of lignin degrading waste water, it is characterized in that, comprise the steps:
The preparation of (1) six kind of bacteria log cell in vegetative period: picking edaphic bacillus (Agrobacteriumsp.) respectively, rod bacterium (Bacillussp.), enterobacter cloacae (Enterobacter.Cloacae.), Gordonia bronchialis (Gordoniasp.), pseudomonas putida (Pseudomonasputida.), Pseudomonas stutzeri (Pseudomonasstutzeri.) six kinds of bacterium 1 ~ 2 rings, it is transferred to respectively containing in 20 ~ 50mL nutritive medium, often kind of bacterium is cultivated 1 ~ 2 day under the condition of 27 ~ 35 DEG C, again six kinds of bacterium after cultivation are seeded in the container containing 300 ~ 500mL proliferated culture medium with the ratio of 1:9 ~ 1:12 in thalline and proliferated culture medium volume ratio respectively, different proliferated culture mediums is adopted to cultivate, wherein, edaphic bacillus, the proliferated culture medium of rod bacterium and enterobacter cloacae is extractum carnis 3.0 ~ 4.0g/L, Tryptones 5.0 ~ 6.0g/L, all the other are water, Gordonia bronchialis vegetative multiplication base is glucose 8.0 ~ 10.0g/L, yeast powder 8.0 ~ 10.0g/L, and all the other are water, pseudomonas putida proliferated culture medium is extractum carnis 1.5g ~ 2.0/L, glucose 1.0 ~ 2.0g/L, Tryptones 6.0 ~ 7.0g/L, yeast powder 3.0 ~ 4.0g/L, and all the other are water, the proliferated culture medium of Pseudomonas stutzeri is casein 17.0 ~ 20.0g/L, potassium hydrogen phosphate 2.5 ~ 3.0g/L, glucose 2.0 ~ 3.0g/L, soyflour 3.0 ~ 4.0g/L, sodium-chlor 4.0 ~ 5.0g/L, and all the other are water, then cultivate 1 ~ 2 day under the condition of 27 ~ 35 DEG C, after the centrifugation 10 ~ 15min of 6000 ~ 7000rpm, obtain the logarithmic phase cell of above-mentioned six kinds of thalline respectively, described nutritive medium main component is extractum carnis 1.5 ~ 2.0g/L, glucose 1.0 ~ 2.0g/L, Tryptones 5.5 ~ 6.5g/L, yeast powder 3.0 ~ 4.0g/L, pH6.5 ~ 7.5, and all the other are water,
The mixed bacterial of (2) six kinds of thalline composite: the logarithmic phase cell of described six kinds of thalline is taken out, after phosphate buffered saline buffer washing, get 3 ~ 6% edaphic bacilluss (Agrobacteriumsp.) respectively, 3 ~ 7% rod bacteriums (Bacillussp.), 10 ~ 26% enterobacter cloacaes (Enterobactercloacae), 24 ~ 35% Gordonia bronchialis (Gordonia), 16 ~ 38% pseudomonas putidas (Pseudomonasputida) and 12 ~ 23% Pseudomonas stutzeris (Pseudomonasstutzeri.) mixing; The composite flora of lignin degrading waste water must be used for.
When described composite flora degraded is containing xylogen waste water, the mixed bacteria liquid getting composite flora is added in nutritive medium with the volume ratio of 1:15 ~ 1:30 and carries out cultivation 6 ~ 12h, directly put in the waste water containing xylogen after composite flora is activated, every 1 liter of waste water containing xylogen adds mixed bacteria liquid 1 ~ 2mL; Air Exposure 3 ~ 5d, aeration rate is 2 ~ 4L/h.
Described nutritive medium main component is extractum carnis 1.5 ~ 2.0g/L, glucose 1.0 ~ 2.0g/L, Tryptones 5.5 ~ 6.5g/L, yeast powder 3.0 ~ 4.0g/L, pH6.5 ~ 7.5, and all the other are water.
By volume percentages, the composition of described phosphate buffered saline buffer is sodium-chlor 8.0 ~ 9.0g/L, Repone K 0.2 ~ 0.3g/L, dipotassium hydrogen phosphate 1.1 ~ 1.2g/L and potassium primary phosphate 0.2 ~ 0.3g/L, and all the other are water.The washing times of described phosphate buffered saline buffer is 2 ~ 3 times.
For a composite flora for lignin degrading waste water, obtained by above-mentioned preparation method.
In described step (3), xylogen waste water is homemade simulated wastewater.Because the present invention is mainly for the exploitation of the degradation flora of xylogen in paper waste, simultaneously in order to better study the degraded situation of composite flora to xylogen, therefore in the present embodiment, adopt homemade simulated wastewater.Above-mentioned homemade xylogen simulated wastewater its preparation method is as follows: the xylogen accurately taking 200 ~ 500mg joins in the aseptic minimal medium of 1L, by pH regulator to 6.0 ~ 9.0 after stirring 30 ~ 60min.The solution wherein preparing waste water is aseptic minimal medium, and its main component is CaCl
20.01 ~ 0.02g/L, KH
2pO
42.0 ~ 2.5g/L, MgSO
40.25 ~ 0.30g/L, ammonium tartrate 0.5 ~ 0.6g/L, NH
4nO
30.5 ~ 0.6g/L, all the other are water.In above-mentioned obtained waste water, the concentration of xylogen is 200 ~ 500mg/L, pH6.0 ~ 9.0.
Tool of the present invention has the following advantages:
1) provided by the inventionly edaphic bacillus (Agrobacteriumsp.) is utilized, rod bacterium (Bacillussp.), enterobacter cloacae (Enterobacter.Cloacae.), Gordonia bronchialis (Gordoniasp.), pseudomonas putida (Pseudomonasputida.), Pseudomonas stutzeri (Pseudomonasstutzeri.) six kinds of bacterium form superior complex strain by a certain percentage and add the biological reinforced process carrying out waste water: pseudomonas putida is often used in wastewater treatment, it has the ability of degraded arene such as toluene and phenol, these are all the common contaminant in municipal wastewater, rod bacterium is a kind of common soil bacteria and aromatic hydrocarbon can be made to produce secondary degraded, and has certain Degradation to the aromatic series such as Pentachlorophenol, Poly Brominated Diphenyl Ethers organic pollutant, Gordonia bronchialis is a kind of oil degradation bacteria, and it can by n-hexadecane, benzene, naphthalene, anthracene, luxuriant and rich with fragrance as carbon source and energy derive, and edaphic bacillus separates bacterial classification from soil, all there is certain Degradation for pollutents such as the most organic pollutant in soil and heavy metals.The present invention finds that the degradation effect of composite flora is far above the single bacterial strain of any strain, illustrates and produces certain interaction between different microorganisms.This due to many biological activities be that individual plant Institute of Micro-biology can not complete or can only faintly carry out, must realize by the interaction of two or more microorganisms in same environment.Single flora and genetic engineering bacterium is adopted to carry out in contaminant degradation process, usually because the generation of inhibition mesostate or intermediate product enter cut-off type meta-bolites approach (endproductpathways) and inhibit contaminant degradation enzymic activity, such that contaminant degradation efficiency is not high or degraded is not thorough.Utilize interaction useful between microorganism, by microorganism mixed culture and domestication, bacterium is flowed to the mesostate of pollutent and carries out some improvement, make inhibition intermediate product not generate or transform as early as possible, thus improve contaminant degradation efficiency.Therefore, utilize microbial interaction, the mixt bacteria microorganism culturing method for domesticating adopted in the practical application of microorganism has more significance.
2) the present invention finds can produce nicotinic acid hydroxylated enzyme and ring opening dioxygenase etc. from by the edaphic bacillus separated the soil of electronics refuse pollution and rod bacterium, Pseudomonas stutzeri then can produce wooden equal hexichol for Yi Xi ?α ， β ?the peroxidase such as dioxygenase.These enzymes can attack simultaneously ehter bond in lignin structure, C ?C key and C
α?C
βdouble bond, makes lignin structure be destroyed rapidly, generates the materials such as the lower aromatics (as Vanillin, vanillic acid and quinones substance) of point quantum, muconate and part small carboxylic acid molecules (formic acid).These organic acid generations can make the pH value of system reduce gradually, thus suppress the growth metabolism of above-mentioned three kinds of bacterium, cause enzymic activity to reduce.The small molecular organic acid that the pseudomonas putida of adding, enterobacter cloacae and Gordonia bronchialis then can utilize lignin degradation to generate fast carries out growth metabolism, and be degraded into the inorganics such as carbonic acid gas and water further, the pH value of system is restored, thus restraining effect is eliminated.Due to the complementation between variant bacterial strain and synergy, their combined action just have very high degradation rate to xylogen.
Accompanying drawing explanation
Fig. 1 is the growing state figure that in embodiment 1, six kinds of thalline are cultivated at aseptic proliferated culture medium.
Fig. 2 is the growing state figure that in embodiment 2, six kinds of thalline are cultivated at aseptic proliferated culture medium.
Fig. 3 is the growing state figure that in embodiment 3, six kinds of thalline are cultivated at aseptic proliferated culture medium.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further illustrated, but the scope of protection of present invention is not limited to the scope of embodiment statement.
Embodiment 1:
Picking edaphic bacillus (Agrobacteriumsp.) respectively, rod bacterium (Bacillussp.), enterobacteria (Enterobacter.Cloacae.), Gordonia bronchialis (Gordoniasp.), pseudomonas putida (Pseudomonasputida.), Pseudomonas stutzeri (Pseudomonasstutzeri.) (from the mud of the water port in paper mill be separated obtain) six kinds of bacterium 1 rings: it is transferred to respectively containing 20mL nutritive medium that (its composition is 1.5g/L extractum carnis, 1.0g/L glucose, 5.5g/L Tryptones, 3.0g/L yeast powder, pH6.5, all the other are water) container in, often kind of bacterium is cultivated 2 days under the condition of 27 DEG C, again the volume ratio (thalline and proliferated culture medium) of six kinds of bacterium difference 1:9 after cultivation is seeded in the container containing 300mL proliferated culture medium, cultivate 2 days under the condition of 27 DEG C, final with after the centrifugation 15min of 6000rpm, obtain the logarithmic phase cell of above-mentioned six kinds of thalline respectively, after normal saline flushing, dry refrigeration is for subsequent use.Wherein, six kinds of different bacteriums adopt different proliferated culture mediums, and the nutritional medium of edaphic bacillus, rod bacterium and enterobacter cloacae is extractum carnis 3.0g/L, Tryptones 5.0g/L; Gordonia bronchialis nutritional medium is glucose 8.0g/L, yeast powder 8.0g/L, and all the other are water; Pseudomonas putida nutritional medium is extractum carnis 1.5g/L, glucose 1.0g/L, Tryptones 6.0g/L, yeast powder 3.0g/L, and all the other are water; The nutritional medium of Pseudomonas stutzeri is casein 17.0g/L, potassium hydrogen phosphate 2.5g/L, glucose 2.0g/L, soyflour 3.0g/L, sodium-chlor 4.0g/L, and all the other are water.The growing state of the rear six kinds of bacteriums of suitable cultivation as shown in Figure 1.As can be seen from Figure 1, the growth curve of six kinds of bacteriums all obviously presents lag phase, logarithmic phase and stationary phase, but different strain reaches the Time Inconsistency needed for each period.Edaphic bacillus (Agrobacteriumsp.) enters logarithmic phase at cultivation 4h, stationary phase is entered after 20h, rod bacterium (Bacillussp.) enters logarithmic phase at cultivation 2h, stationary phase is entered after 20h, enterobacteria (Enterobacter.Cloacae.) enters logarithmic phase at cultivation 1h, stationary phase is entered after 4h, Gordonia bronchialis (Gordonia) enters logarithmic phase at cultivation 1h, just stationary phase is entered after 7h, pseudomonas putida (Pseudomonasputida) just enters logarithmic phase after cultivation 6h, 12h enters stationary phase, Pseudomonas stutzeri (Pseudomonasstutzeri.) just enters logarithmic phase after cultivation 2h, 7h enters stationary phase.
The logarithmic phase cell of above-mentioned six kinds of thalline is taken out, with phosphate buffered saline buffer (its main component sodium-chlor 8.0g/L, Repone K 0.2g/L, dipotassium hydrogen phosphate 1.1g/L and potassium primary phosphate 0.2g/L, all the other are water) wash 2 times after, by volume percentages, get 4% edaphic bacillus (Agrobacteriumsp.) respectively, 6% rod bacterium (Bacillussp.), 18% enterobacter cloacae (Enterobactercloacae), 28% Gordonia bronchialis (Gordonia), 31% pseudomonas putida (Pseudomonasputida) and 13% Pseudomonas stutzeri (Pseudomonasstutzeri.) mixing, and be suspended in physiological saline, refrigerate for subsequent use.
Get mixed bacteria liquid obtained above to add with the volume ratio of 1:20 (mixed bacteria liquid and nutritive medium) and carry out cultivation 6h to aseptic minimal medium, directly put into 1L after making it activate containing in the waste water of xylogen, Air Exposure 5d, aeration rate is 2L/h.In mass concentration, the composition of nutritive medium is 1.5g/L extractum carnis, 1.0g/L glucose, 5.5g/L Tryptones, 3.0g/L yeast powder, pH6.5, and all the other are water.
Because the present invention is mainly for the exploitation of the degradation flora of xylogen in paper waste, simultaneously in order to better study the degraded situation of composite flora to xylogen, therefore in the present embodiment, adopt homemade simulated wastewater.Above-mentioned homemade xylogen simulated wastewater its preparation method is as follows: the xylogen accurately taking 200mg joins in the aseptic minimal medium of 1L, by pH regulator to 6.0 after stirring 30min.The solution wherein preparing waste water is aseptic minimal medium, and its main component is CaCl
20.01g/L, KH
2pO
42.0g/L, MgSO
40.25g/L, ammonium tartrate 0.5g/L, NH
4nO
30.5g/L, all the other are water.In obtained waste water, the concentration of xylogen is 200mg/L, pH6.0.
Content of lignin in the present embodiment method process water is adopted to be in the simulated wastewater of 200mg/L, after Air Exposure 5d, lignin removing rate reaches 85%, apparently higher than the control group 7% not adding composite flora, simultaneously also higher than adding single culture 57%(Agrobacteriumsp.), 37%(Bacillussp.), 26%(Enterobactercloacae), 43%(Gordonia), 39%(Pseudomonasputida) and 58%(Pseudomonasputida), show that composite flora has good degradation effect to xylogen, and be obviously better than single culture.
Embodiment 2
Picking six kinds of bacterium 2 rings respectively: edaphic bacillus (Agrobacteriumsp.), rod bacterium (Bacillussp.), enterobacteria (Enterobacter.Cloacae.), Gordonia bronchialis (Gordoniasp.), pseudomonas putida (Pseudomonasputida.), it is transferred to containing 30mL nutritive medium that (its composition is 2.0g/L extractum carnis by Pseudomonas stutzeri (Pseudomonasstutzeri.) (from the mud of the water port in paper mill be separated obtain) respectively, 1.5g/L glucose, 6.5g/L Tryptones, 3.5g/L yeast powder, pH7.5, all the other are water) container in, often kind of bacterium is cultivated 2 days under the condition of 30 DEG C, again six kinds of bacterium after cultivation are seeded in the container containing 500mL proliferated culture medium with the volume ratio of 1:10 (thalline and nutritive medium) respectively, cultivate 1 day under the condition of 35 DEG C, final with after the centrifugation 10min of 7000rpm, obtain the logarithmic phase cell of above-mentioned six kinds of thalline respectively, after normal saline flushing, dry refrigeration is for subsequent use.Wherein, six kinds of different bacteriums adopt different proliferated culture mediums, and the nutritional medium of edaphic bacillus, rod bacterium and enterobacter cloacae is extractum carnis 4.0g/L, Tryptones 5.5g/L, and all the other are water; Gordonia bronchialis nutritional medium is glucose 9.0g/L, yeast powder 10.0g/L, and all the other are water; Pseudomonas putida nutritional medium is extractum carnis 2.0g/L, glucose 1.5g/L, Tryptones 7.0g/L, yeast powder 3.5g/L, and all the other are water; The nutritional medium of Pseudomonas stutzeri is casein 20.0g/L, potassium hydrogen phosphate 2.7g/L, glucose 3.0g/L, soyflour 3.5g/L, sodium-chlor 5.0g/L, and all the other are water.The growing state of the rear six kinds of bacteriums of suitable cultivation as shown in Figure 2.As can be seen from Figure 2, the growth curve of six kinds of bacteriums all obviously presents lag phase, logarithmic phase and stationary phase, but different strain reaches the Time Inconsistency needed for each period.Edaphic bacillus (Agrobacteriumsp.) enters logarithmic phase at cultivation 2h, stationary phase is entered after 15h, rod bacterium (Bacillussp.) enters logarithmic phase at cultivation 2h, stationary phase is entered after 18h, enterobacteria (Enterobacter.Cloacae.) enters logarithmic phase at cultivation 1h, stationary phase is entered after 6h, Gordonia bronchialis (Gordonia) enters logarithmic phase at cultivation 1h, just stationary phase is entered after 5h, pseudomonas putida (Pseudomonasputida) just enters logarithmic phase after cultivation 3h, 14h enters stationary phase, Pseudomonas stutzeri (Pseudomonasstutzeri.) just enters logarithmic phase after cultivation 5h, 12h enters stationary phase.
The logarithmic phase cell of above-mentioned six kinds of thalline is taken out, with phosphate buffered saline buffer (its main component sodium-chlor 9.0g/L, Repone K 0.25g/L, dipotassium hydrogen phosphate 1.2g/L and potassium primary phosphate 0.25g/L) wash 3 times after, by volume percentages, get 5% edaphic bacillus (Agrobacteriumsp.) respectively, 6% rod bacterium (Bacillussp.), 18% enterobacter cloacae (Enterobactercloacae), 30% Gordonia bronchialis (Gordonia), 18% pseudomonas putida (Pseudomonasputida) and 23% Pseudomonas stutzeri (Pseudomonasstutzeri.) mix and are suspended in physiological saline, refrigerate for subsequent use.
Get mixed bacteria liquid obtained above to add nutritive medium with the volume ratio of 1:15 (mixed bacteria liquid and nutritive medium) and carry out cultivation 8h, directly put into 1L after making it activate containing in the waste water of xylogen, Air Exposure 5d, aeration rate is 4L/h.In mass concentration, the composition of nutritive medium is 2.0g/L extractum carnis, 1.5g/L glucose, 6.5g/L Tryptones, 3.5g/L yeast powder, pH7.5, and all the other are water.
Because the present invention is mainly for the exploitation of the degradation flora of xylogen in paper waste, simultaneously in order to better study the degraded situation of composite flora to xylogen, therefore in the present embodiment, adopt homemade simulated wastewater.Above-mentioned homemade xylogen simulated wastewater its preparation method is as follows: the xylogen accurately taking 500mg joins in the aseptic minimal medium of 1L, by pH regulator to 9.0 after stirring 60min.The solution wherein preparing waste water is aseptic minimal medium, and its main component is CaCl
20.02g/L, KH
2pO
42.5g/L, MgSO
40.27g/L, ammonium tartrate 0.6g/L, NH
4nO
30.6g/L, all the other are water.In obtained waste water, the concentration of xylogen is 500mg/L, pH9.0.
Content of lignin in the present embodiment method process water is adopted to be in the simulated wastewater of 500mg/L, after Air Exposure 5d, lignin removing rate reaches 65%, apparently higher than the control group 6% not adding composite flora, simultaneously also higher than adding single culture 41%(Agrobacteriumsp.), 27%(Bacillussp.), 21%(Enterobactercloacae), 36%(Gordonia), 31%(Pseudomonasputida) and 44%(Pseudomonasputida), show that composite flora has good degradation effect to xylogen, and be obviously better than single culture.
Embodiment 3
Picking six kinds of bacterium 2 rings respectively: edaphic bacillus (Agrobacteriumsp.), rod bacterium (Bacillussp.), enterobacteria (Enterobacter.Cloacae.), Gordonia bronchialis (Gordoniasp.), pseudomonas putida (Pseudomonasputida.), it is transferred to containing 25mL nutritive medium that (its composition is 1.7g/L extractum carnis by Pseudomonas stutzeri (Pseudomonasstutzeri.) (from the mud of the water port in paper mill be separated obtain) respectively, 2.0g/L glucose, 6.0g/L Tryptones, 4.0g/L yeast powder, pH7.0, all the other are water) container in, often kind of bacterium is cultivated 2 days under the condition of 30 DEG C, again six kinds of bacterium after cultivation are seeded in the container containing 400mL proliferated culture medium with the volume ratio of 1:12 (thalline and nutritive medium) respectively, cultivate 2 days under the condition of 35 DEG C, final with after the centrifugation 10min of 7000rpm, obtain the logarithmic phase cell of above-mentioned six kinds of thalline respectively, after normal saline flushing, dry refrigeration is for subsequent use.Wherein, six kinds of different bacteriums adopt different proliferated culture mediums, and wherein the nutritional medium of edaphic bacillus, rod bacterium and enterobacter cloacae is extractum carnis 3.5g/L, Tryptones 6.0g/L, and all the other are water; Gordonia bronchialis nutritional medium is glucose 10.0g/L, yeast powder 9.0g/L, and all the other are water; Pseudomonas putida nutritional medium is extractum carnis 1.7g/L, glucose 2.0g/L, Tryptones 6.5g/L, yeast powder 4.0g/L, and all the other are water; The nutritional medium of Pseudomonas stutzeri is casein 19.0g/L, potassium hydrogen phosphate 3.0g/L, glucose 2.5g/L, soyflour 4.0g/L, sodium-chlor 4.5g/L, and all the other are water.The growing state of the rear six kinds of bacteriums of suitable cultivation as shown in Figure 3.As can be seen from Figure 3, the growth curve of six kinds of bacteriums all obviously presents lag phase, logarithmic phase and stationary phase, but different strain reaches the Time Inconsistency needed for each period.Edaphic bacillus (Agrobacteriumsp.) enters logarithmic phase at cultivation 5h, stationary phase is entered after 18h, rod bacterium (Bacillussp.) enters logarithmic phase at cultivation 2h, stationary phase is entered after 20h, enterobacteria (Enterobacter.cloacae.) enters logarithmic phase at cultivation 1h, stationary phase is entered after 7h, Gordonia bronchialis (Gordonia) enters logarithmic phase at cultivation 1h, just stationary phase is entered after 8h, pseudomonas putida (Pseudomonasputida) just enters logarithmic phase after cultivation 6h, 14h enters stationary phase, Pseudomonas stutzeri (Pseudomonasstutzeri.) just enters logarithmic phase after cultivation 2h, 10h enters stationary phase.
The logarithmic phase cell of above-mentioned six kinds of thalline is taken out, with phosphate buffered saline buffer, (its main component is sodium-chlor 8.5g/L, Repone K 0.3g/L, dipotassium hydrogen phosphate 1.15g/L and potassium primary phosphate 0.3g/L, all the other are water) wash 3 times after, by volume percentages, get 3% edaphic bacillus (Agrobacteriumsp.) respectively, 6% rod bacterium (Bacillussp.), 16% enterobacter cloacae (Enterobactercloacae), 26% Gordonia bronchialis (Gordonia), 26% pseudomonas putida (Pseudomonasputida) and 23% Pseudomonas stutzeri (Pseudomonasstutzeri.) mix and are suspended in physiological saline, refrigerate for subsequent use.
Get mixed bacteria liquid obtained above to add with the volume ratio of 1:20 (mixed bacteria liquid and nutritive medium) and carry out cultivation 12h to nutritive medium, directly put into 1L after making it activate containing in the waste water of xylogen, Air Exposure 4d, aeration rate is 3L/h.In mass concentration, nutrient composition is 1.7g/L extractum carnis, 2.0g/L glucose, 6.0g/L Tryptones, 4.0g/L yeast powder, pH7.0, and all the other are water.
Because the present invention is mainly for the exploitation of the degradation flora of xylogen in paper waste, simultaneously in order to better study the degraded situation of composite flora to xylogen, therefore in the present embodiment, adopt homemade simulated wastewater.Above-mentioned homemade xylogen simulated wastewater its preparation method is as follows: the xylogen accurately taking 400mg joins in the aseptic minimal medium of 1L, by pH regulator to 8.0 after stirring 50min.The solution wherein preparing waste water is aseptic minimal medium, and its main component is CaCl
20.015g/L, KH
2pO
42.3g/L, MgSO
40.30g/L, ammonium tartrate 0.55g/L, NH
4nO
30.55g/L, all the other are water.In obtained waste water, the concentration of xylogen is 400mg/L, pH8.0.
Content of lignin in the present embodiment method process water is adopted to be in the simulated wastewater of 400mg/L, after Air Exposure 4d, lignin removing rate reaches 75%, apparently higher than the control group 64% not adding composite flora, simultaneously also higher than adding single culture 47%(Agrobacteriumsp.), 29%(Bacillussp.), 21%(Enterobactercloacae), 39%(Gordonia), 34%(Pseudomonasputida) and 50%(Pseudomonasputida), show that composite flora has good degradation effect to xylogen, and be obviously better than single culture.
In the present invention, the microflora formed by optimum combination can realize the collaborative and complementary action of six kinds of microorganisms in system, overcome in single culture lignin degrading process the problem being easily subject to mesostate and suppressing, make whole system be all have efficient degradation capability (65 ~ 85%) to lower concentration or the waste water of the xylogen of high density.Report according to Related Bacteria lignin degrading: Rhodopseudomonas is as the most effective lignin degradation person, it is less than 10% [KamodaS to the degradation efficiency of xylogen, SaburiY.Structuralandenzymaticalcomparisonoflignostilben e ?α β ?dioxygenaseisozymes, I, II, andIII, fromPseudomonaspaucimobilisTMY1009.BioscienceBiotechnolo gy & Biochemistry, 1993,57 (6): 931 ?934, CarolAC.BacterialAssociationswithDecayingWood:aReview.In ternationalBiodetertoration & Biodegradation, 1996, 37 (1): 101 ?107], and the most high degradation rate of filamentous bacterium to lower xylogen belonging to streptomycete also only has 20% [GoddenB, BallAS, HelvensteinP, etal.Towardselucidationofthelignindegradationpathwayinac tinomycetes.JournalofGeneralandAppliedMicrobiology, 1992, 138:2441 ?2448].The degradation efficiency of these single bacteriums to xylogen is starkly lower than composite flora of the present invention, and therefore this composite flora has a good application prospect in process xylogen waste water.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1. for a preparation method for the composite flora of lignin degrading waste water, it is characterized in that, comprise the steps:
The preparation of (1) six kind of bacteria log cell in vegetative period: picking edaphic bacillus (Agrobacteriumsp.) respectively, genus bacillus (Bacillussp.), enterobacter cloacae (Enterobactercloacae), Gordonia bronchialis (Gordonaspp.), pseudomonas putida (Pseudomonasputida) and Pseudomonas stutzeri (Pseudomonasstutzeri) six kinds of bacterium 1 ~ 2 rings, it is transferred to respectively containing in 20 ~ 50mL nutritive medium, often kind of bacterium is cultivated 1 ~ 2 day under the condition of 27 ~ 35 DEG C, again six kinds of bacterium after cultivation are seeded in the container containing 300 ~ 500mL proliferated culture medium with the ratio of 1:9 ~ 1:12 in thalline and proliferated culture medium volume ratio respectively, different proliferated culture mediums is adopted to cultivate, wherein, edaphic bacillus, the proliferated culture medium of rod bacterium and enterobacter cloacae is extractum carnis 3.0 ~ 4.0g/L, Tryptones 5.0 ~ 6.0g/L, all the other are water, Gordonia bronchialis proliferated culture medium is glucose 8.0 ~ 10.0g/L, yeast powder 8.0 ~ 10.0g/L, and all the other are water, pseudomonas putida proliferated culture medium is extractum carnis 1.5g ~ 2.0/L, glucose 1.0 ~ 2.0g/L, Tryptones 6.0 ~ 7.0g/L, yeast powder 3.0 ~ 4.0g/L, and all the other are water, the proliferated culture medium of Pseudomonas stutzeri is casein 17.0 ~ 20.0g/L, potassium hydrogen phosphate 2.5 ~ 3.0g/L, glucose 2.0 ~ 3.0g/L, soyflour 3.0 ~ 4.0g/L, sodium-chlor 4.0 ~ 5.0g/L, and all the other are water, then cultivate 1 ~ 2 day under the condition of 27 ~ 35 DEG C, after the centrifugation 10 ~ 15min of 6000 ~ 7000rpm, obtain the logarithmic phase cell of above-mentioned six kinds of thalline respectively, described nutritive medium main component is extractum carnis 1.5 ~ 2.0g/L, glucose 1.0 ~ 2.0g/L, Tryptones 5.5 ~ 6.5g/L, yeast powder 3.0 ~ 4.0g/L, pH6.5 ~ 7.5, and all the other are water,
The mixed bacterial of (2) six kinds of thalline composite: the logarithmic phase cell of described six kinds of thalline is taken out, after phosphate buffered saline buffer washing, get 3 ~ 6% edaphic bacilluss (Agrobacteriumsp.) respectively, 3 ~ 7% genus bacillus (Bacillussp.), 10 ~ 26% enterobacter cloacaes (Enterobactercloacae), 24 ~ 35% Gordonia bronchialis (Gordonaspp.), 16 ~ 38% pseudomonas putidas (Pseudomonasputida) and 12 ~ 23% Pseudomonas stutzeris (Pseudomonasstutzeri) mixing; The composite flora of lignin degrading waste water must be used for;
When described composite flora degraded is containing xylogen waste water, the mixed bacteria liquid getting composite flora is added in nutritive medium with the volume ratio of 1:15 ~ 1:30 and carries out cultivation 6 ~ 12h, directly put in the waste water containing xylogen after composite flora is activated, every 1 liter of waste water containing xylogen adds mixed bacteria liquid 1 ~ 2mL; Air Exposure 3 ~ 5d, aeration rate is 2 ~ 4L/h.
2. the preparation method of the composite flora for lignin degrading waste water according to claim 1, it is characterized in that, by volume percentages, the composition of described phosphate buffered saline buffer is sodium-chlor 8.0 ~ 9.0g/L, Repone K 0.2 ~ 0.3g/L, dipotassium hydrogen phosphate 1.1 ~ 1.2g/L and potassium primary phosphate 0.2 ~ 0.3g/L, all the other are water.
3. the preparation method of the composite flora for lignin degrading waste water according to claim 1 and 2, is characterized in that, the washing times of described phosphate buffered saline buffer is 2 ~ 3 times.
4. for a composite flora for lignin degrading waste water, it is characterized in that, obtained by preparation method described in claim 1 or 2.
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