CN104845905A - High-efficiency composite inocula for removing salad oil-containing waste water COD and application thereof - Google Patents
High-efficiency composite inocula for removing salad oil-containing waste water COD and application thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 claims abstract description 66
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses a high-efficiency composite inocula for removing salad oil-containing waste water COD and an application thereof. The composite inocula is composed of four high efficiency grease degrading bacteria, and are respectively candida tropicalis HY-A-3, serratia plymuthica SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2. According to the invention, a bacterium source sample is collected from activated sludge and restaurant sewer sewage in a sewage treatment factory, single bacterium capable of removing salad oil waste water COD with high efficiency can be screened by separating; then the high efficiency composite inocula is constructed through a complex formulation experiments, and the result is characterized in that an inocula combination of HY-A-3, SH-AB-1, SZ2-A-3 and LB1-AB-2 has the highest COD clearance rate. When the concentration of the composite inocula is 1-5%, the inocula combination has more than 75% on salad oil waste water COD clearance. The inocula has good application prospect and value in during salad oil waste water processing engineering.
Description
Technical field
The present invention relates to a kind of high efficiency composition microbial inoculum removed containing salad oil wastewater COD, also relate to the composite fungus agent application method removing salad oil wastewater COD.
Background technology
Along with salad oil demand sharply rises, the highly concentrated oil-bearing wastewater in salad oil processing causes concern on the impact that environment causes.Salad oil manufacturing wastewater refer to the squeezing such as soybean or the crude oil that leaches gained are come unstuck, depickling, decolouring, deodorization, dewaxing, de-ester etc. process the waste water produced.Produce a large amount of organic waste water in this refining process, the feature of this waste water is high COD (ρ
cODcan more than 10000mg/L be reached), the large (ρ of oil-contg
oilcan more than 3000mg/L be reached), B/C 0.5-0.6.At present physico-chemical process pre-treatment oil removing and activated sludge process or anaerobic technique process are adopted more for the process of salad oil manufacturing wastewater.After amount of grease enters Sewage treatment systems, usually can cause producing bubble, and hyphomycetic growth can be promoted, affect the operation of sewage disposal biochemical system.Therefore be the deoiling function of the biochemical system in strengthening salad oil wastewater treating processes, accelerate the degraded to grease, the possibility that the water outlet of reduction system exceeds standard, reduce the foam produced, core---the enhancement microbiological of salad oil wastewater process provided by the invention, utilize the functional flora screening yeast and the bacterium formation obtained from occurring in nature, in aerobic biological treatment process, efficient-decomposition is carried out to the lipid acid of high density, glyceryl ester and other organism, to realize the effluent quality improving Sewage treatment systems.
Summary of the invention
For defect of the prior art, the object of this invention is to provide composite fungus agent of a kind of efficient removal salad oil wastewater COD and preparation method thereof.
Another object of the present invention is to provide the application method of above-mentioned composite fungus agent.
In order to realize foregoing invention object, the technical solution used in the present invention is as follows:
Remove the composite fungus agent containing salad oil wastewater COD, it is characterized in that, described composite fungus agent comprises bacterial strain: candida tropicalis HY-A-3, CGMCC No.10424; Serratia SH-AB-1, CGMCCNo.10427; Candida tropicalis SZ2-A-3, CGMCC No.10425; Serratia marcescens LB1-AB-2, CGMCC No.10426.
Further, the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 25-45%, 25-45%, 8-25% and 2-25% of total bacterium amount volume respectively.
Further, the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 30-40%, 30-40%, 15-20%, 3-20% of total bacterium amount volume respectively.
Further, the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 33%, 33%, 17%, 17% of total bacterium amount volume respectively.
Present invention also offers a kind of preparation method removing the composite fungus agent containing salad oil wastewater COD, comprise the following steps: get the mono-colony inoculation of candiyeast HY-A-3 and SZ2-A-3 respectively in 100mL yeast extract powder peptone dextrose culture-medium, get Serratia SH-AB-1 and the mono-colony inoculation of serratia marcescens LB1-AB-2 in 100mL beef extract-peptone liquid nutrient medium, at 180rpm, shaking culture 18h at 28 DEG C; The four strain bacterium cultivating 18h are carried out compound.
Present invention also offers a kind of method removed containing salad oil wastewater COD, comprise the steps: the composite fungus agent comprising candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 to be inoculated in salad oil wastewater by the proportioning of 25-45%, 25-45%, 8-25% and 2-25% of accounting for total bacterium amount volume respectively, and cultivate 2 ~ 3 days.
Further, the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 30-40%, 30-40%, 15-20% and 3-20% of total bacterium amount volume respectively.
Further, the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 33%, 33%, 17% and 17% of total bacterium amount volume respectively.
Further, described composite fungus agent is with the inoculation of bacterium liquid form, and its inoculum size is the 1-5% (v/v) of process wastewater volume.
Further, described inoculum size is the 1.5-2.5% (v/v) of process wastewater volume.
Effect of the present invention is: adopt composite fungus agent of the present invention and application thereof, can reach more than 75% to the clearance containing salad oil wastewater COD.
Accompanying drawing explanation
The systematic evolution tree of Fig. 1 candida tropicalis Candida tropicalis HY-A-3;
The systematic evolution tree of Fig. 2 Serratia Serratia sp.SH-AB-1;
The systematic evolution tree of Fig. 3 candida tropicalis Candida tropicalis SZ2-A-3;
The systematic evolution tree of Fig. 4 serratia marcescens Serratia marcescens LB1-AB-2.
Classification And Nomenclature: candida tropicalis, Latin name: Candida tropicalis HY-A-3
Preservation date: on January 22nd, 2015;
Depositary institution's full name and abbreviation: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Deposit number: CGMCC No.10424;
Classification And Nomenclature: Serratia, Latin name: Serratia sp.SH-AB-1
Preservation date: on January 22nd, 2015;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Deposit number: CGMCC No.10427;
Classification And Nomenclature: candida tropicalis, Latin name: Candida tropicalis SZ2-A-3
Preservation date: on January 22nd, 2015;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Deposit number: CGMCC No.10425;
Classification And Nomenclature: serratia marcescens, Latin name: Serratia marcescens LB1-AB-2
Preservation date: on January 22nd, 2015;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Deposit number: CGMCC No.10426;
Embodiment
Below illustrate the present invention, but the protection domain be not intended to limit the present invention.
In order to solve the problem, the present invention gathers the samples such as certain dining room, shopping center effluent sewerage, other lower ditch, market of farm produce meat stand sewage, sewage work's biochemical reaction tank mud and the other body refuse of certain vegetables oil source mill treatment tank, through enrichment, domestication, separation, purifying, filter out the pure bacterial strain having and remove salad oil wastewater COD, again through primary dcreening operation, multiple sieve, select efficient function yeast, build the composite fungus agent removing salad oil wastewater COD.
HY-A-3 in the present invention is located away from certain dining room, shopping center effluent sewerage; SH-AB-1 is located away from the sewage of other lower ditch, meat stand, the market of farm produce, Beijing; SZ2-A-3 is located away from Hebei sewage work biochemical reaction tank mud; LB1-AB-2 is located away from the other body refuse of Beijing vegetables oil source mill treatment tank.Morphological specificity observation and the qualification of part Physiology and biochemistry are carried out respectively to 4 strain bacterium in composite fungus agent.HY-A-3 thalli morphology ovalize, size is about 5 ~ 6 × 7 ~ 9 μm,, oyster white, smooth, protruding, neat in edge rounded at the upper bacterium colony of yeast extract powder peptone dextrose culture-medium (YPD substratum), still can better grow in the NaCl solution of 10%; SH-AB-1 thalline is rod-short, and size is about 0.5 ~ 0.8 × 0.9 ~ 1.3 μm, without gemma, bacterium colony is rounded, protruding, smooth, oyster white, Gram-negative, and catalase is positive, Starch Hydrolysis is negative, can not grow in the NaCl solution more than 7% concentration; SZ2-A-3 thalli morphology ovalize, size is about 5 ~ 7 × 8 ~ 9 μm, circular, smooth, protruding, the oyster white of bacterium colony on YPD substratum, and catalase is positive, still can better grow in the NaCl solution of 10%; LB1-AB-2 thalline is rod-short, and size is about 0.5 ~ 0.8 × 0.6 ~ 1.0 μm, and without gemma, bacterium colony is circular, smooth, protruding, red, Gram-negative, and catalase is positive, and Starch Hydrolysis is negative, can not grow in the NaCl solution more than 7% concentration.Carry out molecular biology identification respectively to 4 strain bacterium, HY-A-3 and SZ2-A-3 carries out the pcr amplification of 18S rDNA after extracting gene DNA, SH-AB-1 and LB1-AB-2 carries out the pcr amplification of 16S rDNA, the fragment of acquisition is used for order-checking.After order-checking, sequence results carries out Blast comparison in Genbank, and constructing system evolutionary tree, as Figure 1-Figure 4.
By the systematic evolution tree of sequencing result and structure, in conjunction with 4 strain bacterium morphological specificitys, growth conditions, Physiology and biochemistry qualification result, can preliminary evaluation bacterial strain HY-A-3 and SZ2-A-3 be candida tropicalis (Candidatropicalis), LB1-AB-2 is that serratia marcescens (Serratia marcescens), SH-AB-1 belong to serratia (Serratia sp.).Above-mentioned four kinds of bacterial strains are all deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City).
In composite fungus agent of the present invention, the bacterium amount of candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3, serratia marcescens LB1-AB-2 accounts for 25-45%, 25-45%, 8-25% and 2-25% of total bacterium amount volume respectively, preferably accounts for 30-40%, 30-40%, 15-20% and 3-20% of total bacterium amount volume respectively.
The preparation method of above-mentioned composite fungus agent is, get the mono-colony inoculation of candiyeast HY-A-3 and SZ2-A-3 respectively in 100mL yeast extract powder peptone dextrose culture-medium (YPD liquid nutrient medium), get Serratia SH-AB-1 and the mono-colony inoculation of serratia marcescens LB1-AB-2 in 100mL beef extract-peptone liquid nutrient medium, at 180rpm, shaking culture 18h at 28 DEG C.Candida tropicalis HY-A-3, the Serratia SH-AB-1, candida tropicalis SZ2-A-3, the serratia marcescens LB1-AB-2 that cultivate 18h are carried out compound by bacterium amount than 30-40%, 30-40%, 15-20% and the 3-20% accounting for total bacterium amount volume.
The application of above-mentioned composite fungus agent, is inoculated in containing salad oil mass by composite fungus agent by the concentration of 1-5% and is the oil wastewater of 1000-2000mg/L, can reaches more than 75% to the clearance of waste water COD.
Embodiment 1: the acclimating of grease degrading strain and separation screening.
First the present invention gathers rich grease-contained bacterium source, many places sample, then carries out acclimating cultivation to bacterium source sample respectively, more therefrom isolates pure bacterial strain, then obtains high-efficiency grease degradation bacteria through primary dcreening operation, multiple sieve.
Concrete implementation step is as follows:
(1) acclimating of grease degrading strain is cultivated and initial gross separation
Get 3g mud sample or 3mL water sample, be inoculated in the triangular flask containing 100mL enrichment culture liquid, and 28 DEG C, cultivate 3d in the shaking table of 180rpm.Get the bacterium liquid after 3mL enrichment, be inoculated in and tame in the triangular flask of nutrient solution containing 100mL, 28 DEG C, 180rpm shaking table is cultivated, and 3d is 1 cycle, is moved to by bacterium liquid on new domestication substratum and cultivate after each cycle terminates.
Get the bacterium liquid after 1mL domestication, be inoculated in respectively containing in 100mL bacterium or yeast liquid basal medium, be placed in 28 DEG C, the shaking table of 180rpm is cultivated.After 3d, after the bacterium liquid gradient dilution of cultivation, coat in corresponding solid-based basal culture medium flat board respectively, cultivate 48-72h at 30 DEG C of incubators.The microorganism strains of picking colony morphological specificity significant difference, streak inoculation is in the basic medium of correspondence respectively, 30 DEG C of constant temperature culture 48-72h, and obtain single bacterium colony pure growth through repeating line separation, inclined-plane is preserved.
(2) screening of high-efficiency grease degradation bacteria
Line is separated the bacterial strain point obtained to receive on lipase checking culture medium flat plate, 30 DEG C of constant incubators cultivate 48-72h, just sift out the bacterial strain that transparent circle diameter is larger, for next step multiple sieve provides foundation.
With colony inoculation in transfering loop picking primary dcreening operation flat board in the triangular flask containing 100mL basic medium, 28 DEG C, the cultivation of 180rpm shaking table, transfer in the triangular flask containing 100mL salad oil wastewater by 2% inoculum size after cultivating 18h, 28 DEG C, 180rpm shaker fermentation, respectively at 48h, 72h sampling and measuring COD.Last combining fat enzyme circle proof test, obtains the stronger bacterial strain of 8 strain degraded grease ability.
In the present invention, required medium preparing is as follows:
1. enrichment medium: K
2hPO
43g, NaCl 5g, MgSO
47H
2o 0.5g, peptone 10g, yeast extract paste 5g, salad oil 10g, distilled water 1000mL, pH=7.2.
2. substratum is tamed: yeast extract paste 1g, (NH
4)
2sO
43g, K
2hPO
41g, NaH
2pO
41g, NaCl0.5g, MgSO
47H
2o 0.2g, salad oil adds 5g, 10g, 15g successively, distilled water 1000mL, pH7.0.
3. lipase checking substratum: KH
2pO
40.3g, MgSO
47H
2o 0.1g, K
2hPO
41.5g, (NH
4)
2sO
41g, NaCl 5g, FeSO
47H
2o 0.01g, Tween 80 20mL, salad oil 6g, agar 18g, distilled water 1000mL, pH7.0.
4. autogamy oily(waste)water: KH
2pO
40.3g, MgSO
47H
2o 0.1g, K
2hPO
41.5g, (NH
4)
2sO
41g, NaCl 5g, FeSO
47H
2o 0.01g, salad oil 2g, distilled water 1000mL, pH7.0.
Embodiment 2: the structure of composite fungus agent in the present invention.
Filter out 8 plant height effect grease degrading strains, combine experiment is carried out in choosing wherein 4 strains, and in often kind of combination, 4 kinds of bacterium bacterium amount ratios are identical, be inoculated in autogamy oily(waste)water, measure the COD of salad oil wastewater after 72h by total inoculum size 2%.The combination of what COD clearance was the highest be combined as HY-A-3, SH-AB-1, SZ2-A-3 and LB1-AB-2, its COD clearance is 80.18%, through qualification, 4 strain bacterium are respectively candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3, serratia marcescens LB1-AB-2.In aforesaid combination 4 kind of bacterium is carried out to four factor three horizontal quadrature tests of different vaccination ratio.Research shows, between each bacterial strain, best inoculation volume proportion HY-A-3:SH-A-1:SZ2-A-3:LB1-AB-2 is 33%, 33%, 17% and 17%, the total bacterium amount of mix bacterium agent is inoculated in oily(waste)water by the inoculative proportion of 2%, and after 72h, oil wastewater COD clearance is 81.3% (former water COD1426.2mg/L).
Embodiment 3: be with above-described embodiment difference, 25%, 25%, 25% and 25% proportioning that candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 tetra-strain bacterium account for total bacterium amount volume by bacterium amount is respectively carried out compound, mix bacterium agent is seeded in oily(waste)water in the ratio of 1%, after 72h, oil wastewater COD clearance is 75.98% (former water COD1076.32mg/L).
Embodiment 4: be with above-described embodiment difference, 45%, 45%, 8% and 2% proportioning that candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 tetra-strain bacterium account for total bacterium amount volume by bacterium amount is respectively carried out compound, mix bacterium agent is seeded in oily(waste)water in the ratio of 5%, after 72h, oil wastewater COD clearance is 80.15% (former water COD1058.49mg/L).
Embodiment 5: in the present invention, composite fungus agent is tested actual oil wastewater COD degradation.
Actual waste water takes from certain Peasants Joy food and drink waste water, and crossed two-layer gauze removing dish leaf leftovers before using, the initial COD of waste water is 1723.6mg/L, pH is 6.0.Get the actual oily(waste)water of 100mL in 250mL triangular flask, after regulating pH to 7.2, by 2% inoculum size inoculating compound bacterium agent, setting does not inoculate the oily(waste)water of microbial inoculum in contrast, with being placed in 180rpm, 28 DEG C of condition bottom fermentations cultivations, measures COD after 72h.The clearance of composite fungus agent to actual oily(waste)water COD is 78.95%.
It should be noted that; above-mentioned specific embodiment is only exemplary; under above-mentioned instruction of the present invention, those skilled in the art can carry out various improvement and distortion on the basis of above-described embodiment, and these improve or distortion drops in protection scope of the present invention.It will be understood by those skilled in the art that specific descriptions are above to explain object of the present invention, not for limiting the present invention.Protection scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. remove the composite fungus agent containing salad oil wastewater COD, it is characterized in that, described composite fungus agent comprises bacterial strain: candida tropicalis HY-A-3, CGMCC No.10424; Serratia SH-AB-1, CGMCCNo.10427; Candida tropicalis SZ2-A-3, CGMCC No.10425; Serratia marcescens LB1-AB-2, CGMCC No.10426.
2. a kind of composite fungus agent removed containing salad oil wastewater COD according to claim 1, is characterized in that: the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 25-45%, 25-45%, 8-25% and 2-25% of total bacterium amount volume respectively.
3. a kind of composite fungus agent removed containing salad oil wastewater COD according to claim 2, is characterized in that: the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 30-40%, 30-40%, 15-20% and 3-20% of total bacterium amount volume respectively.
4. a kind of composite fungus agent removed containing salad oil wastewater COD according to claim 3, is characterized in that: the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 33%, 33%, 17% and 17% of total bacterium amount volume respectively.
5. the preparation method of the composite fungus agent described in the arbitrary claim of claim 1-4, comprise the following steps: get the mono-colony inoculation of candiyeast HY-A-3 and SZ2-A-3 respectively in 100mL yeast extract powder peptone dextrose culture-medium, get Serratia SH-AB-1 and the mono-colony inoculation of serratia marcescens LB1-AB-2 in 100mL beef extract-peptone liquid nutrient medium, at 180rpm, shaking culture 18h at 28 DEG C; The four strain bacterium cultivating 18h are carried out compound.
6. remove the method containing salad oil wastewater COD for one kind, comprise the steps: the composite fungus agent comprising candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 to be inoculated in salad oil wastewater by 25-45%, 25-45%, 8-25% and 2-25% proportioning accounting for total bacterium amount volume respectively, and cultivate 2 ~ 3 days.
7. a kind of method removed containing salad oil wastewater COD according to claim 6, is characterized in that: the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 30-40%, 30-40%, 15-20% and 3-20% of total bacterium amount volume respectively.
8. a kind of method removed containing salad oil wastewater COD as claimed in claim 7, is characterized in that: the bacterium amount of described candida tropicalis HY-A-3, Serratia SH-AB-1, candida tropicalis SZ2-A-3 and serratia marcescens LB1-AB-2 accounts for 33%, 33%, 17% and 17% of total bacterium amount volume respectively.
9. a kind of method removed containing salad oil wastewater COD as described in claim 6,7 or 8, is characterized in that: described composite fungus agent is with the inoculation of bacterium liquid form, and its inoculum size is the 1-5% (v/v) of process wastewater volume.
10. a kind of method removed containing salad oil wastewater COD as claimed in claim 9, is characterized in that: described inoculum size is the 1.5-2.5% (v/v) of process wastewater volume.
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