CN103525720A - Efficient grease degrading bacterium and application thereof - Google Patents
Efficient grease degrading bacterium and application thereof Download PDFInfo
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- CN103525720A CN103525720A CN201310403853.5A CN201310403853A CN103525720A CN 103525720 A CN103525720 A CN 103525720A CN 201310403853 A CN201310403853 A CN 201310403853A CN 103525720 A CN103525720 A CN 103525720A
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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 microorganism fermentative production field, say more specifically a kind of high-efficiency grease degradation bacteria and application thereof.
Background technology
The treatment process of the waste water that lipid is polluted mainly contains three kinds: 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 extensively, need the shortcomings such as specific installation.These class methods can not effectively be processed grease rubbish simultaneously, also have the danger that produces secondary pollution.In contrast to this, if biological treatment rule is to utilize biological know-how microorganism, using grease as the required carbon source of microorganism growth and the energy, grease is shifted and transformed, under the catalysis of enzyme, be hydrolyzed into glycerine, lipid acid, be finally degraded to H
2o, CO
2deng.Compare 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 wide, easy cultivation, reproduction speed is fast, environmental compatibility is strong of originating, so biological treatment is being brought into play increasing effect aspect processing grease rubbish.At present, the processing, washing that Japan, more American-European developed countries have been applied to grease degrading strain biologic garbage degraded, oily(waste)water waits reinforcement with the application in these areas of the multinomial field such as the production of enzyme and biofuel ，Er China.
The biologic treating technique of waste grease has UASB method, biomembrance process, biological contact oxidation process, BAC biologic treating technique, supercritical CO
2decomposition technique etc.In recent years, people have found again the treatment process of various efficient degradation greases, and by making for improving processing efficiency combining of the whole bag of tricks.By add can efficient degradation grease microorganism be undoubtedly one of most suitable method.
There is the microorganism of the grease of much degrading in occurring in nature, especially in the abundant soil and waste water of oleaginousness.The microorganism cutting grease mainly comprises the aerobism kind in bacterium and fungi.In the middle of the degradation bacteria of having 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 in fungi (Candida), Rhodotorula (Rhodotorula), Trichosporon (Trichosporon), Geotrichum (Geotrichum), Yarrowia belongs to, Sporobolomyces (Sporobolomyces) etc.At present existing many for mixed bacterial in the research aspect grease degraded, and that screening and the bacterial strain that obtains high-efficiency grease degradation capability are still is one of crucial.
Simultaneously bio-surfactant is the surface-active meta-bolites that has of the class set hydrophilic group that produced under certain culture condition by microorganism and hydrophobic group and one.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, the present bio-surfactant of having developed many excellent performances, the applied industrial circle of bio-surfactant is also in continuous expansion.Bio-surfactant is mainly divided into two classes: non-ionic type and anionic, and 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 is along with the different preparation methods of microorganism also vary, thereby is 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, zymotechnique and the cost-effective recovery method of design high productivity, oneself becomes the focus of people's research.Bio-surfactant is compared and is still occupied larger inferior position with synthetic tensio-active agent, is first that the extraction separation of bio-surfactant is more complicated, has increased production cost.And the lower concentration of bio-surfactant in fermented liquid and amphipathic normal obstruction it is 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 degradation bacteria, high-efficiency grease degradation bacteria is by acinetobacter calcoaceticus UC13; Wherein, acinetobacter calcoaceticus UC13 is in June, 2013 24 China Committee for Culture Collection of Microorganisms common micro-organisms center preservation, 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 processing; And the application of high-efficiency grease degradation bacteria in the relevant industry refining of grease.Described grease is vegetables oil, animal oil or mineral oil.
, bio-surfactant is to take fermentation culture acinetobacter calcoaceticus UC13 products therefrom in the fermention medium that peanut oil is sole carbon source.
It is described that to take the fermentation culture based component that peanut oil is sole carbon source be 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, take acinetobacter calcoaceticus UC13 as producing bacterial strain, cultivates by fermentation successively, fermented liquid is centrifugal, regulate supernatant liquor pH value, organic solvent extraction, organic phase are dry, obtain tensio-active agent after decompress filter, underpressure distillation.
Concrete steps are:
(1) fermentation: be seeded in and take in the fermention medium that peanut oil is sole carbon source by the inoculum size that adds 1-2ml UC13 bacterial strain in every 100ml substratum; 100-120h is cultivated in speed oscillation with 140-160r/min under the constant temperature of 28-30 ℃;
(2) centrifugal: to get fermented liquid at 8000-10000r/min, centrifugal 10-15min at 4-8 ℃;
(3) adjust pH: centrifugal rear collection supernatant liquor, then regulate pH value to 2.0-3.0 supernatant liquor;
(4) extraction: the supernatant liquor that mixes up pH is extracted with ethyl acetate, and the volume ratio of ethyl acetate and supernatant liquor is 1:1-1:2;
(5) dry: (mass volume ratio) Na that adds 6-8% in the organic phase after extraction
2sO
4dry 6-10h;
(6) filter: remove Na
2sO
4precipitation;
(7) distillation: underpressure distillation is removed ethyl acetate and obtained tensio-active agent at 40-45 ℃.
Describedly with the HCl of 4-6mol/L, regulate pH value to 2.0-3.0 centrifugal gained supernatant liquor.
It is described that to take the fermentation culture based component that peanut oil is sole carbon source be 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 of the changing food waste removing commercial grease and contain grease.Tensio-active agent is apparent is light yellow soup compound.There is good emulsification and oil extraction performance.
Acinetobacter calcoaceticus UC13 preserves center preservation on June 24th, 2013 in Chinese microorganism, 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 ℃), the degradation rate of the soybean oil that 24h is 10mg/mL to concentration, peanut oil, lard can reach respectively 60%, 64%, 50%.
The present invention has advantages of: bio-surfactant of the present invention is to obtain the active product of tensio-active agent preferably through a strain grease degrading strain fermentation.Utilize extracting method of the present invention, the extraction of carrying out bio-surfactant that can be simple and quick, the bio-surfactant of gained is light yellow soup compound, 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 grease degradation capability analysis chart that Fig. 2 provides for the embodiment of the present invention.
The affect figure of the temperature that Fig. 3 provides for the embodiment of the present invention on UC13 grease degradation capability.
The affect figure of the pH that Fig. 4 provides for the embodiment of the present invention on UC13 grease degradation capability
Fig. 5 tests design sketch for generation and the emulsifying property of the tensio-active agent that the embodiment of the present invention provides, and 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 to this.
Embodiment 1: bacterial strain acclimating and separation and Culture
The mud sample 5-10g that gathers Yantai City, Shandong Yi Jia food processing plant grease treating pond, adds in the 150-300mL triangular flask that 50-100mL sterilized water and sterile glass beads are housed, and is placed on shaking table, the 150-180r/min 10-15min that fully vibrates, 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, water is settled to 1000mL) in, 28-30 ℃, 150-250r/min cultivates.Every 24h observes grease degraded and dissipation situation, adds in right amount peanut oil to 4ml.Every 4-6d switching 5-10mL culture is to the new substratum of 30-50mL, to promote the growth of grease decomposer.This domestication process continues 2 months.
Prepare grease selectivity dull and stereotyped, get respectively Tryptones 10g, yeast extract 5g, NaCl10g, agar powder 12g, peanut oil 10g, 1.6%(mass volume ratio: g/ml) neutral red aqueous solution 1mL, with distilled water, be settled to 1L, add 2-3%(mass volume ratio: agarose g/ml).121 ℃ of high-temperature sterilization 30min.Get the bacterium liquid that appropriate above-mentioned domestication obtains, through water, be diluted to 10
-3-10
-6after be coated on above-mentioned flat board.Selection has the bacterium colony of obvious grease degraded circle, separated through repeating line, obtains the pure growth of single bacterium colony, after name, preserves.Through 16s rDNA, analyze, determine that UC13 is a strain acinetobacter calcoaceticus (Acinetobacter sp.).
Embodiment 2: grease degrading activity is analyzed
By after UC13 bacterial strain activation, be seeded in and take in the substratum that peanut oil is sole carbon source, 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 ℃, rotating speed 150-250r/min; Get respectively and be cultured to the 0th, the bacterium liquid of 6,12,24,30,36,42,48h.Measure respectively above-mentioned each bacterium liquid light absorption value under 600nm condition and determine the increment of bacterium; Each 5mL of fermented liquid that simultaneously gets respectively the different fermentations time, uses respectively isopyknic n-hexane extraction twice, merges organic phase.Organic phase after merging dewaters dry by anhydrous sodium sulphate, be placed in round-bottomed flask, 40-45 ℃ of decompress filter, and resistates is the quality of the rear residue of degraded grease.Degradation rate=(quality (mg) of 50-residue grease)/50 * 100%.As shown in Figure 1.Along with the growth of bacterium, in fermented liquid, the degraded of grease is obviously strengthened.
The degradation capability of embodiment 3:UC13 to different greases
By after UC13 bacterial strain activation, be seeded in respectively and take in the substratum that peanut oil, soybean oil, sesame oil, lard, diesel oil is sole carbon source.Substratum, except fat type difference, is same as the composition of embodiment 2.By above-mentioned fermented liquid fermentation culture in constant-temperature shaking incubator, culture temperature is 28-30 ℃, rotating speed 150-250r/min respectively; Be cultured to the bacterium liquid of 24h.Measure 600nm light absorption value and determine the increment of bacterium; Get respectively 5mL fermented liquid, use n-hexane extraction.According to the method for embodiment 2, calculate degradation rate.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, for the degradation capability of plant grease, be slightly better than animal quasi-grease.For industrial diesel oil, there is certain degradation capability simultaneously.
Embodiment 4: the impact of temperature on UC13 degradation capability
By after UC13 bacterial strain activation, according to 1%(v/v) inoculum size be seeded in and take in the substratum that peanut oil is sole carbon source.Medium component is same as embodiment 2.Fermented liquid is divided into three parts, is placed in respectively the shaking table of 25,30,37 ℃, rotating speed 150-250r/min cultivates; Above-mentioned fermented liquid is cultivated to 48h, get respectively 5mL fermented liquid, use n-hexane extraction.According to the method for embodiment 2, calculate degradation rate.Temperature on the impact of UC13 degraded grease as shown in Figure 3.25-30 ℃ of degraded that is conducive to grease.
The impact of embodiment 5:pH on UC13 degradation capability
By after UC13 bacterial strain activation, according to 1%(v/v) inoculum size be seeded in and take in the substratum that peanut oil is sole carbon source.Medium component is same as embodiment 2.Fermented liquid is divided into three parts, and regulating respectively pH is 6,7,8.Fermented liquid is placed in to the shaking table of 28-30 ℃, rotating speed 150-250r/min cultivates; Above-mentioned fermented liquid is cultivated to 48h, get respectively 5mL fermented liquid, use n-hexane extraction.According to the method for embodiment 2, calculate degradation rate.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 is seeded in and take in the fermention medium that peanut oil is sole carbon source; In constant-temperature shaking incubator, carry out fermentation culture; In culturing process, culture temperature is 28-30 ℃, 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 at 8000-10000r/min, centrifugal 15-20min at 4-8 ℃;
3) regulate the pH value of supernatant liquor: collect the supernatant liquor after centrifugal, supernatant liquor by the HCl adjusting pH value of 4-6mol/L to 2.0-3.0;
4) organic solvent extraction: the supernatant liquor that mixes up pH is extracted with ethyl acetate, and 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, obtain without Na
2sO
4the extraction phase of impurity;
7) distillation: underpressure distillation at 40-45 ℃, remove organic solvent and obtain tensio-active agent.The ethyl acetate that distillation obtains is returned to step (5) recycling.
Embodiment 7: emulsifying property analysis
By UC13 bacterial strain according to 1%(volume ratio) inoculum size is seeded in and take in the fermention medium that peanut oil is sole carbon source; In constant-temperature shaking incubator, carry out fermentation culture; In culturing process, culture temperature is 28-30 ℃, 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.
In 15mL or 50mL centrifuge tube, add respectively the above-mentioned UC13 of 5mL to cultivate the nutrient solution after 24h, then add respectively again 5mL whiteruss.Add after paraffin in 80W ultrasonication 30S(referring to Fig. 5 A), standing 24h(is referring to Fig. 5 B at 25 ℃), measure oil phase, the volume of water 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%.
Embodiment 8 degreasing activity analyses
By UC13 bacterial strain according to 1%(volume ratio) inoculum size is seeded in and take in the fermention medium that peanut oil is sole carbon source; In constant-temperature shaking incubator, carry out fermentation culture; In culturing process, culture temperature is 28-30 ℃, 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, at 8000-10000r/min, centrifugal 15-20Min at 4-8 ℃, collects supernatant liquor, stand-by;
The glass culture dish that is 9.5cm by diameter rinses after twice through acid soak, distilled water, the distilled water that adds 30mL, on the water surface, add 0.2mL peanut oil to form with thin oil film, at oil film center, slowly add the above-mentioned supernatant liquor of 0.1mL, 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 (10)
1. a high-efficiency grease degradation bacteria, is characterized in that: high-efficiency grease degradation bacteria is by acinetobacter calcoaceticus UC13; Wherein, acinetobacter calcoaceticus UC13 preserves center preservation on June 24th, 2013 in Chinese microorganism, and deposit number is CGMCC No:7818.
2. an application for high-efficiency grease degradation bacteria claimed in 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 processing; And the application of high-efficiency grease degradation bacteria in the relevant industry refining of grease.
3. by the application of high-efficiency grease degradation bacteria claimed in claim 2, it is characterized in that: described grease is vegetables oil, animal oil or mineral oil.
4. a bio-surfactant, is characterized in that: bio-surfactant is to take fermentation culture acinetobacter calcoaceticus UC13 products therefrom in the fermention medium that peanut oil is sole carbon source.
5. by bio-surfactant claimed in claim 4, it is characterized in that: described to take the fermentation culture based component that peanut oil is sole carbon source be 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.
6. the extracting method of a bio-surfactant claimed in claim 4, it is characterized in that: take acinetobacter calcoaceticus UC13 as producing bacterial strain, cultivate by fermentation successively, fermented liquid is centrifugal, regulate supernatant liquor pH value, organic solvent extraction, organic phase are dry, obtain tensio-active agent after decompress filter, underpressure distillation.
7. by the extracting method of bio-surfactant claimed in claim 6, it is characterized in that: concrete steps are:
(1) fermentation: by UC13 bacterial strain according to 1-2%(v/v) inoculum size is seeded in and take in the fermention medium that peanut oil is sole carbon source; 100-120h is cultivated in speed oscillation with 140-160r/min under the constant temperature of 28-30 ℃;
(2) centrifugal: to get fermented liquid at 8000-10000r/min, centrifugal 10-15min at 4-8 ℃;
(3) adjust pH: centrifugal rear collection supernatant liquor, then regulate pH value to 2.0-3.0 supernatant liquor;
(4) extraction: the supernatant liquor that mixes up pH is extracted with ethyl acetate, and the volume ratio of ethyl acetate and supernatant liquor is 1:1-1:2;
(5) dry: (mass volume ratio) Na that adds 6-8% in the organic phase after extraction
2sO
4dry 6-10h;
(6) filter: remove Na
2sO
4precipitation;
(7) distillation: underpressure distillation is removed ethyl acetate and obtained tensio-active agent at 40-45 ℃.
8. by the extracting method of bio-surfactant claimed in claim 6, it is characterized in that: describedly with the HCl of 4-6mol/L, regulate pH value to 2.0-3.0 centrifugal gained supernatant liquor.
9. by the extracting method of bio-surfactant claimed in claim 6, it is characterized in that: described to take the fermentation culture based component that peanut oil is sole carbon source be 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.
10. an application for bio-surfactant claimed in claim 4, is characterized in that: described bio-surfactant is for the pollutent of the changing food waste removing commercial grease and contain grease.
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