CN103525720B - Efficient grease degrading bacterium and application thereof - Google Patents
Efficient grease degrading bacterium and application thereof Download PDFInfo
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- CN103525720B CN103525720B CN201310403853.5A CN201310403853A CN103525720B CN 103525720 B CN103525720 B CN 103525720B CN 201310403853 A CN201310403853 A CN 201310403853A CN 103525720 B CN103525720 B CN 103525720B
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Abstract
The invention belongs to the production field of microbial fermentation, and in particular relates to an efficient grease degrading bacterium and an application thereof. The efficient grease degrading bacterium is of acinetobacter UC13, wherein the acinetobacter UC13 is preserved in CGMCC (China General Microbiological Culture Collection Center) on June 24th, 2013; the preservation number of the acinetobacter UC13 is CGMCC No:7818. The invention further relates to applications of the efficient grease degrading bacterium in treatment of kitchen wastes or petroleum hydrocarbon contaminants containing greases and in industrial refining relevant to the greases. A biological surfactant is a fermentation product obtained by fermenting the acinetobacter UC13. The biological surfactant is used for eliminating industrial greases and contaminants in the kitchen wastes containing the greases. The invention provides an extraction method of the biological surfactant. The extraction method is simple to operate and can be used for ensuring the high activity of the biological surfactant.
Description
Technical field
The invention belongs to fermentable production field, is a kind of high-efficiency grease degradation bacteria and application thereof in particular.
Background technology
Three kinds are mainly contained to the treatment process of the waste water that lipid pollutes: Physical, chemical method and biological process.The physico-chemical processes such as picture is saltoutd, electrolysis, membrane sepn, have investment large, take up an area wide, need the shortcomings such as specific installation.These class methods can not effectively process grease rubbish simultaneously, also there is the danger producing secondary pollution.In contrast to this, if biological treatment rule utilizes biological know-how microorganism, using grease as the carbon source needed for microorganism growth and the energy, grease shifted and transforms, under the catalysis of enzyme, being hydrolyzed into glycerine, lipid acid, being finally degraded to H
2o, CO
2deng.Compared with physico-chemical process, biological treatment has that cost is low, floor space is few, do not need specific installation, can not bring the advantages such as secondary pollution.And biological treatment mainly relies on microorganism, and microorganism has the advantages such as source extensively, is easily cultivated, reproduction speed is fast, environmental compatibility is strong, and therefore biological treatment plays increasing effect in process grease rubbish.At present, grease degrading strain has been applied to the multinomial field such as production of biologic garbage degraded, the process of oily(waste)water, washing enzyme and biofuel by Japan, more American-European developed countries, and China's application in these areas waits to strengthen.
The biologic treating technique of waste grease has UASB method, biomembrance process, biological contact oxidation process, BAC biologic treating technique, supercritical CO 2 decomposition technique etc.In recent years, people have found again the treatment process of various efficient degradation grease, and improve processing efficiency by the conbined usage of various method.Can microorganism beyond doubt one of most suitable method of efficient degradation grease by adding.
There is the microorganism of grease of much degrading in occurring in nature, especially in the abundant soil of oleaginousness and waste water.The microorganism that cut grease mainly comprises the aerobism kind in bacterium and fungi.In the middle of the degradation bacteria found, bacterium is more, and fungi strain is less.Mainly contain the Pseudomonas (Pseudomonas) in bacterium, Flavobacterium (Flavobacterium), Alkaligenes (Alcaligenes), genus arthrobacter (Arthrobacter), micrococcus sp (Micrococcus), bacillus (Bacillus), Rhodopseudomonas (Rhodopseudomonas), Thiobacillus (Thiobacillus), Nitrobacter (Nitrobacter), Staphylococcus (StapHylococcus) etc., mycocandida (Candida) in fungi, Rhodotorula (Rhodotorula), Trichosporon (Trichosporon), Geotrichum (Geotrichum), Yarrowia belongs to, Sporobolomyces (Sporobolomyces) etc.Existing many for the research of mixed bacterial in oils degradation at present, and the bacterial strain of screening and acquisition high-efficiency grease degradation capability is still one of key.
Simultaneously bio-surfactant be the class set hydrophilic group and hydrophobic group that are produced under certain culture condition by microorganism with integrally there is surface-active meta-bolites.The research of bio-surfactant, not from recent years, has just started the research to bio-surfactant as far back as 20 century 70s.Now, people are no longer strange to bio-surfactant.And developed now the bio-surfactant of many excellent performances, the industrial circle that bio-surfactant is applied is also in continuous expansion.Bio-surfactant is mainly divided into two classes: non-ionic type and anionic, cationic comparatively rare.According to mechanical feature classification, bio-surfactant can be divided into five classes: glycolipid, lipopeptid, polysaccharide protein complex compound, phosphatide and lipid acid or neutral fat.
Bio-surfactant also varies along with the different preparation methods of microorganism, is thus difficult to provide general desirable operation instruction route.In order to obtain large productive rate, transformation efficiency and ultimate density, the superior strain of seed selection bio-surfactant, the zymotechnique of design high productivity and cost-effective recovery method, oneself becomes the focus of people's research.Bio-surfactant still occupies larger inferior position compared with the tensio-active agent of synthesis, is first that the extraction and isolation of bio-surfactant is more complicated, adds production cost.And the lower concentration of bio-surfactant in fermented liquid and amphipathic normal obstruction its be effectively separated.Along with deepening continuously of research, some traditional methods are constantly perfect, and new method constantly occurs.The conventional extraction of bio-surfactant has: solvent extraction, crystallization and precipitation, foam fractionation method, ultrafiltration process, tlc (TLC) etc.
Summary of the invention
The object of the invention is a kind of high-efficiency grease degradation bacteria and application thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of high-efficiency grease degradation bacteria, high-efficiency grease degradation bacteria is by acinetobacter calcoaceticus UC13; Wherein, acinetobacter calcoaceticus UC13 on June 24th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No:7818, taxonomy called after acinetobacter calcoaceticus Acinetobacter sp..
The application of high-efficiency grease degradation bacteria, the application of described high-efficiency grease degradation bacteria in grease-contained changing food waste or petroleum hydrocarbon class pollutant process; And the application of high-efficiency grease degradation bacteria in the industry refining that grease is relevant.Described grease is vegetables oil, animal oil or mineral oil.
A kind of bio-surfactant, bio-surfactant be with peanut oil be sole carbon source fermention medium in fermentation culture acinetobacter calcoaceticus UC13 products therefrom.
Described take peanut oil as the fermentation medium components of sole carbon source is peanut oil 10-20mg/mL, NH
4nO
30.2-0.5g/L, K
2hPO
40.5-1.5g/L, KH
2pO
40.5-1.5g/L, MgSO
4.7H
2o0.1-0.5g/L.
The extracting method of bio-surfactant, with acinetobacter calcoaceticus UC13 for producing bacterial strain, cultivating by fermentation successively, fermented liquid be centrifugal, regulate supernatant liquor pH value, organic solvent extraction, organic phase drying, decompress filter, underpressure distillation after obtain tensio-active agent.
Concrete steps are:
(1) ferment: be seeded in by the inoculum size adding 1-2ml UC13 bacterial strain in every 100ml substratum with peanut oil be sole carbon source fermention medium in; 100-120h is cultivated with the speed oscillation of 140-160r/min under the constant temperature of 28-30 DEG C;
(2) centrifugal: to get fermented liquid centrifugal 10-15min at 8000-10000r/min, 4-8 DEG C;
(3) adjust pH: collected after centrifugation supernatant liquor, then by supernatant liquor adjust ph to 2.0-3.0;
(4) extract: be extracted with ethyl acetate by the supernatant liquor mixing up pH, the volume ratio of ethyl acetate and supernatant liquor is 1:1-1:2;
(5) dry: (mass volume ratio) Na adding 6-8% in organic phase after the extraction
2sO
4dry 6-10h;
(6) filter: remove Na
2sO
4precipitation;
(7) distill: at 40-45 DEG C, underpressure distillation removing ethyl acetate obtains tensio-active agent.
The described HCl by centrifugal gained supernatant liquor 4-6mol/L carries out adjust ph to 2.0-3.0.
Described take peanut oil as the fermentation medium components of sole carbon source is peanut oil 10-20mg/mL, NH
4nO
30.2-0.5g/L, K
2hPO
40.5-1.5g/L, KH
2pO
40.5-1.5g/L, MgSO
4.7H
2o0.1-1.5g/L.
The application of bio-surfactant, described bio-surfactant is for the pollutent removing commercial grease and contain in the changing food waste of grease.Tensio-active agent is apparent is light yellow syrup.There is good emulsification and oil extraction performance.
Acinetobacter calcoaceticus UC13 preserves center preservation on June 24th, 2013 at China Microbiological, and deposit number is CGMCC No:7818.This bacterium all has certain degradation capability to soybean oil, peanut oil, lard, diesel oil.Under room temperature condition (25-30 DEG C), 24h to concentration be the soybean oil of 10mg/mL, the degradation rate of peanut oil, lard can reach 60%, 64%, 50% respectively.
The advantage that the present invention has: bio-surfactant of the present invention is the product obtaining active tensio-active agent preferably through a strain grease degrading strain fermentation.Utilize extracting method of the present invention, the extraction carrying out bio-surfactant that can be simple and quick, the bio-surfactant of gained is light yellow syrup, and the output of thick product is 10-15g/L.And there is good emulsifying property and degreasing activity.
Accompanying drawing explanation
The growth of the UC13 that Fig. 1 provides for the embodiment of the present invention and peanut oil degraded situation map.
The UC13 oils degradation capability analysis figure that Fig. 2 provides for the embodiment of the present invention.
The temperature that Fig. 3 provides for the embodiment of the present invention is to the effect diagram of UC13 oils degradation ability.
The pH that Fig. 4 provides for the embodiment of the present invention is to the effect diagram of UC13 oils degradation ability
The generation of the tensio-active agent that Fig. 5 provides for the embodiment of the present invention and emulsifying property test design sketch, wherein A is the sample after supersound process; B is sample after standing 24h.
The oil extraction performance test design sketch that Fig. 6 provides for the embodiment of the present invention.
Embodiment
The embodiment of carrying out around bacterial strain of the present invention is below described in detail, but embodiments of the present invention are not limited thereto.
Embodiment 1: bacterial strain acclimating and separation and Culture
Gather the mud sample 5-10g of the food processing plant's grease treating pond in Shandong Yantai City, add and be equipped with in the 150-300mL triangular flask of 50-100mL sterilized water and sterile glass beads, be placed on shaking table, 150-180r/min fully vibrates 10-15min, get mixed solution 5-8mL, add aseptic enrichment culture liquid (NaCl0.5, the peptone 5.0 of 35-50mL, extractum carnis 0.5, peanut oil 4mL, pH7.2-7.4, be settled to 1000mL with water) in, 28-30 DEG C, 150-250r/min cultivate.Every 24h observes oils degradation and dissipation situation, adds peanut oil in right amount to 4ml.Every 4-6d transfers 5-10mL culture in the new substratum of 30-50mL, to promote the growth of grease decomposer.This domestication process lasts 2 months.
Prepare grease selectivity dull and stereotyped, get Tryptones 10g respectively, yeast extract 5g, NaCl10g, agar powder 12g, peanut oil 10g, 1.6%(mass volume ratio: g/ml) neutral red aqueous solution 1mL, be settled to 1L with distilled water, add 2-3%(mass volume ratio: g/ml) agarose.121 DEG C of high-temperature sterilization 30min.Get the bacterium liquid that appropriate above-mentioned domestication obtains, be diluted to 10 through water
-3-10
-6after be coated on above-mentioned flat board.Select the bacterium colony with obvious oils degradation circle, being separated through repeating line, obtaining the pure growth of single bacterium colony, preserving after name.Analyze through 16s rDNA, determine that UC13 is a strain acinetobacter calcoaceticus (Acinetobacter sp.).
Embodiment 2: oils degradation activation analysis
By UC13 bacterial strain activate after, be seeded in peanut oil be sole carbon source substratum in, medium component is peanut oil 10mg/mL, NH
4nO
30.2g/L, K
2hPO
40.5g/L, KH
2pO
40.5g/L, MgSO
4.7H
2o0.1g/L; Fermentation culture in constant-temperature shaking incubator, culture temperature is 28-30 DEG C, rotating speed 150-250r/min; Get the bacterium liquid being cultured to the 0th, 6,12,24,30,36,42,48h respectively.Measure above-mentioned each bacterium liquid light absorption value under 600nm condition respectively and determine the increment of bacterium; Get each 5mL of fermented liquid of different fermentations time simultaneously respectively, respectively with isopyknic n-hexane extraction twice, merge organic phase.Organic phase anhydrous sodium sulphate after merging dewaters drying, is placed in round-bottomed flask, 40-45 DEG C of decompress filter, and resistates is the quality of residue grease after degraded.Degradation rate=(50-remains the quality (mg) of grease)/50 × 100%.As shown in Figure 1.Along with the growth of bacterium, in fermented liquid, the degraded of grease is obviously strengthened.
Embodiment 3:UC13 is to the degradation capability of different grease
After UC13 bacterial strain is activated, be seeded in respectively with in peanut oil, soybean oil, sesame oil, lard, the diesel oil substratum that is sole carbon source.Substratum, except fat type difference, is same as the composition of embodiment 2.Respectively by above-mentioned fermented liquid fermentation culture in constant-temperature shaking incubator, culture temperature is 28-30 DEG C, rotating speed 150-250r/min; Be cultured to the bacterium liquid of 24h.Measure the increment that 600nm light absorption value determines bacterium; Get 5mL fermented liquid respectively, use n-hexane extraction.Degradation rate is calculated according to the method for embodiment 2.UC13 to the degraded situation of surveyed different greases as shown in Figure 2.UC13 all has degradation capability to surveyed grease.Degradation capability for edible oil is better than technical oils.In edible oil, the degradation capability for plant grease is slightly better than animal quasi-grease.For industrial diesel oil, there is certain degradation capability simultaneously.
Embodiment 4: temperature is on the impact of UC13 degradation capability
By UC13 bacterial strain activate after, according to 1%(v/v) inoculum size be seeded in peanut oil be sole carbon source substratum in.Medium component is same as embodiment 2.Fermented liquid is divided into three parts, is placed in the shaking table of 25,30,37 DEG C respectively, rotating speed 150-250r/min cultivates; Above-mentioned fermented liquid is cultivated 48h, gets 5mL fermented liquid respectively, use n-hexane extraction.Degradation rate is calculated according to the method for embodiment 2.Temperature on the impact of UC13 degraded grease as shown in Figure 3.The 25-30 DEG C of degraded being conducive to grease.
Embodiment 5:pH is on the impact of UC13 degradation capability
By UC13 bacterial strain activate after, according to 1%(v/v) inoculum size be seeded in peanut oil be sole carbon source substratum in.Medium component is same as embodiment 2.Fermented liquid is divided into three parts, regulates pH to be 6,7,8 respectively.Fermented liquid is placed in the shaking table of 28-30 DEG C, rotating speed 150-250r/min cultivates; Above-mentioned fermented liquid is cultivated 48h, gets 5mL fermented liquid respectively, use n-hexane extraction.Degradation rate is calculated according to the method for embodiment 2.PH on the impact of UC13 degraded grease as shown in Figure 4.Alkaline condition is conducive to the degraded of grease.
Embodiment 6: the extraction of tensio-active agent
1) by UC13 bacterial strain according to 1%(volume ratio) inoculum size be seeded in peanut oil be sole carbon source fermention medium in; Fermentation culture is carried out in constant-temperature shaking incubator; In culturing process, culture temperature is 28-30 DEG C, and incubation time is 96-120h; Constant temperature oscillation case rotating speed is 150-250r/min; Medium component is peanut oil 10-20mg/mL, NH
4nO
30.2g/L, K
2hPO
40.5g/L, KH
2pO
40.5g/L, MgSO
4.7H
2o0.1g/L;
2) supernatant liquor is centrifugal: by the above-mentioned fermented liquid through fermentation culture centrifugal 15-20min at 8000-10000r/min, 4-8 DEG C;
3) regulate the pH value of supernatant liquor: collect centrifugal after supernatant liquor, the HCl adjust ph of supernatant liquor 4-6mol/L is to 2.0-3.0;
4) organic solvent extraction: the supernatant liquor mixing up pH is extracted with ethyl acetate, the volume ratio of ethyl acetate and supernatant liquor is 1-1.2:1;
5) organic phase is dry: merge the organic phase after extraction, add (mass volume ratio, g/ml) Na of 5-8%
2sO
4dry 1.5-2h; .
6) filter: dried organic phase is filtered, obtains without Na
2sO
4the extraction phase of impurity;
7) distill: underpressure distillation at 40-45 DEG C, removing organic solvent obtains tensio-active agent.The ethyl acetate that distillation obtains returns step (5) recycling.
Embodiment 7: emulsifying property analysis
By UC13 bacterial strain according to 1%(volume ratio) inoculum size be seeded in peanut oil be sole carbon source fermention medium in; Fermentation culture is carried out in constant-temperature shaking incubator; In culturing process, culture temperature is 28-30 DEG C, and incubation time is 48h; Constant temperature oscillation case rotating speed is 150-250r/min; Medium component is peanut oil 10-20mg/mL, NH
4nO
30.2g/L, K
2hPO
40.5g/L, KH
2pO
40.5g/L, MgSO
4.7H
2o0.1g/L.
Add the nutrient solution after 5mL above-mentioned UC13 cultivation 24h in 15mL or 50mL centrifuge tube respectively, then add 5mL whiteruss respectively again.To add after paraffin in 80W ultrasonication 30S(see Fig. 5 A), leave standstill 24h(at 25 DEG C see Fig. 5 B), measure oil phase, the volume of aqueous phase and Emulsion Phase, represents emulsifying capacity (%) with the volume of Emulsion Phase and the ratio of cumulative volume.As shown in Figure 1.The emulsifying capacity of this tensio-active agent can reach 50%-55%.
The analysis of embodiment 8 degreasing activity
By UC13 bacterial strain according to 1%(volume ratio) inoculum size be seeded in peanut oil be sole carbon source fermention medium in; Fermentation culture is carried out in constant-temperature shaking incubator; In culturing process, culture temperature is 28-30 DEG C, and incubation time is 48-96h; Constant temperature oscillation case rotating speed is 150-250r/min; Medium component is peanut oil 10-20mg/mL, NH
4nO
30.2g/L, K
2hPO
40.5g/L, KH
2pO
40.5g/L, MgSO
4.7H
2o0.1g/L.By the above-mentioned fermented liquid through fermentation culture centrifugal 15-20Min at 8000-10000r/min, 4-8 DEG C, collect supernatant liquor, stand-by;
Be that the glass culture dish of 9.5cm is after acid soak, distilled water rinse twice by diameter, add the distilled water of 30mL, the water surface adds 0.2mL peanut oil to be formed with thin oil film, the above-mentioned supernatant liquor of 0.1mL is slowly added at oil film center, center oil film is pressed against surrounding and forms a circle, and circle diameter is approximately directly proportional to the content of tensio-active agent.(Fig. 6).
Claims (7)
1. a high-efficiency grease degradation bacteria, is characterized in that: high-efficiency grease degradation bacteria is acinetobacter calcoaceticus UC13Acinetobacter; Wherein, acinetobacter calcoaceticus UC13 on June 24th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No:7818.
2. an application for high-efficiency grease degradation bacteria according to claim 1, is characterized in that: the application of described high-efficiency grease degradation bacteria in grease-contained changing food waste or petroleum hydrocarbon class pollutant process; And the application of high-efficiency grease degradation bacteria in the industry refining that grease is relevant;
Described grease is vegetables oil, animal oil or mineral oil.
3. a bio-surfactant, is characterized in that: bio-surfactant be with peanut oil be sole carbon source fermention medium in fermentation culture acinetobacter calcoaceticus UC13 according to claim 1 products therefrom; With acinetobacter calcoaceticus UC13 for producing bacterial strain, cultivate by fermentation successively, fermented liquid be centrifugal, regulate supernatant liquor pH value, organic solvent extraction, organic phase drying, decompress filter, underpressure distillation after obtain tensio-active agent, described take peanut oil as the fermentation medium components of sole carbon source is peanut oil 10-20mg/mL, NH
4nO
30.2-0.5g/L, K
2hPO
40.5-1.5g/L, KH
2pO
40.5-1.5g/L, MgSO
4.7H
2o0.1-1.5g/L.
4. the extracting method of a bio-surfactant according to claim 3, it is characterized in that: with acinetobacter calcoaceticus UC13 for producing bacterial strain, cultivate by fermentation successively, fermented liquid be centrifugal, regulate supernatant liquor pH value, organic solvent extraction, organic phase drying, decompress filter, underpressure distillation after obtain tensio-active agent, described take peanut oil as the fermentation medium components of sole carbon source is peanut oil 10-20mg/mL, NH
4nO
30.2-0.5g/L, K
2hPO
40.5-1.5g/L, KH
2pO
40.5-1.5g/L, MgSO
4.7H
2o0.1-1.5g/L.
5., by the extracting method of bio-surfactant according to claim 4, it is characterized in that: concrete steps are:
(1) ferment: UC13 bacterial strain is seeded according to 1-2% (v/v) inoculum size with peanut oil be sole carbon source fermention medium in; 100-120h is cultivated with the speed oscillation of 140-160r/min under the constant temperature of 28-30 DEG C;
(2) centrifugal: to get fermented liquid centrifugal 10-15min at 8000-10000r/min, 4-8 DEG C;
(3) adjust pH: collected after centrifugation supernatant liquor, then by supernatant liquor adjust ph to 2.0-3.0;
(4) extract: be extracted with ethyl acetate by the supernatant liquor mixing up pH, the volume ratio of ethyl acetate and supernatant liquor is 1:1-1:2;
(5) dry: (mass volume ratio) Na adding 6-8% in organic phase after the extraction
2sO
4dry 6-10h;
(6) filter: remove Na
2sO
4precipitation;
(7) distill: at 40-45 DEG C, underpressure distillation removing ethyl acetate obtains tensio-active agent.
6., by the extracting method of bio-surfactant according to claim 4, it is characterized in that: the described HCl by centrifugal gained supernatant liquor 4-6mol/L carries out adjust ph to 2.0-3.0.
7. an application for bio-surfactant according to claim 3, is characterized in that: described bio-surfactant is for the pollutent removing commercial grease and contain in the changing food waste of grease.
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| CN110438033B (en) * | 2019-07-02 | 2021-04-27 | 浙江工业大学 | Grease degrading bacterium, application and grease degrading method |
| CN110283757B (en) * | 2019-07-25 | 2022-06-10 | 常州大学 | Grease degrading bacterium IUMR B53 and application thereof |
| CN110283759B (en) * | 2019-07-25 | 2022-05-17 | 常州大学 | Separation and application of grease degrading bacterium Kosakonia cowaniiuMR B67 |
| CN110283758B (en) * | 2019-07-25 | 2022-06-10 | 常州大学 | Grease degrading bacterium IUMR B55 and application thereof |
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