CN105695444A - Immobilized microbial inoculant for ship repairing and building sewage treatment, and preparation method and application thereof - Google Patents
Immobilized microbial inoculant for ship repairing and building sewage treatment, and preparation method and application thereof Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
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Abstract
The invention relates to an immobilized microbial inoculant for ship repairing and building sewage treatment. The microbial inoculant is characterized by comprising a halophilic petroleum hydrocarbon degrading bacterium and an immobilization material. The immobilization material comprises sodium alginate, CaCl2, mussel shell powder and 2-5 wt% citric acid, wherein the mass ratio of the mussel shell powder to the citric acid is 1:(1-3), and the mass ratio of the mussel shell powder to the sodium alginate to the CaCl2 is 1:(0.8-1):(0.7-0.8). The preparation method of the immobilized microbial inoculant is simple and convenient, and is controllable in operation. The prepared immobilized microbial inoculant can be easily preserved and reutilized; the degradation rate for oil pollutants in ship repairing and building wastewater can reach 82-94%, and is enhanced by 39-47% as compared with free microbes; and the environment tolerance is enhanced.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of immobilized microorganism microbial inoculum for Ship Building and Repairing sewage disposal and its preparation method and application。
Background technology
World Port industry, shipping industry fast development, promoted the further flourishing of Ship Building and Repairing industry, along with World Ship repairs the east shifting of industry, provided good opportunity to develop to the development of Chinese shipbuilding industry。While the marine economy such as Ship Building and Repairing industry and near port industry develop, boats and ships washing of tanks and repair procedures build sewage by substantial amounts of for generation, building sewage and include oily waste water and sanitary sewage, wherein oily waste water includes again cabin oily waste water, water ballast, dock bottom oily waste water etc.。Building sewage and have a strong impact on local marine eco-environment safety, region is built sewage and is rationally processed, comprehensively utilizes the great environmental issue becoming Development of Marine economy and near port industry, the protection marine eco-environment。
Ship Building and Repairing is produced saliferous and carrying out a biological disposal upon the developing direction being to build sewage disposal at present containing the waste water of greasy dirt, build in Sewage Biological Treatment and be capable of withstanding the activated sludge by certain salinity range thus except carrying out a biological disposal upon except being obtained by sludge acclimatization, from hypersaline environment around, enrichment culture screening addicted to salt petroleum hydrocarbon degradation bacterium or chooses applicable strain the biological treatment for Ship Building and Repairing waste water from existing strain, is also one method fast and effectively。
The method can be that efficiently processing of Ship Building and Repairing sea of retribution salt oily water provides good microorganism resource, but in strain use procedure, tradition occupation mode is all liquid microbial preparation, and liquid microbial preparation exists problems such as being easily washed out reactor and cause timeliness short under current flow regime。
Summary of the invention
First to be solved by this invention technical problem is that and provide a kind of immobilized microorganism microbial inoculum for Ship Building and Repairing sewage disposal for prior art。
The preparation method that second technical problem to be solved by this invention is to provide a kind of above-mentioned immobilized microorganism microbial inoculum。
3rd technical problem to be solved by this invention is to provide a kind of above-mentioned immobilized microorganism microbial inoculum in the application in Ship Building and Repairing sewage disposal。
This invention address that the technical scheme that first technical problem adopts is: a kind of immobilized microorganism microbial inoculum for Ship Building and Repairing sewage disposal, it is characterized in that, including addicted to salt petroleum hydrocarbon degradation thalline and immobilization material, this immobilization material includes sodium alginate, CaCl2, mussel shell powder and concentration be the citric acid of 2～5wt%, wherein mussel shell powder is 1:1～3 with the mass ratio of citric acid, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8～1:0.7～0.8。
As preferably, described immobilized microorganism microbial inoculum is microspheroidal, and wherein the concentration addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107～2 × 108cell/g。
As preferably, described addicted to salt petroleum hydrocarbon degradation thalline by Rhodopseudomonas Pseudomonas, pimelobacter sp belong to Pimelobacter, sea Coccus Marinococcus, Microbacterium Microbacterium and Planococcus Planococcus form, mass ratio is 3～5:1～3:2～3:1～2:1～2。
Further, heretofore described Rhodopseudomonas is B-1, antibacterial respectively N1, N2, N3 and N4 of described pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus, and obtain each through following steps: 25 DEG C of activation in enrichment medium will be accessed containing bacterium source water sample, bacterium solution after activation is injected in crude oil culture medium, 25 DEG C, 180～200r/min cultivate domestication, finally with oil flat board carry out repeatedly separate, purification obtain N1～N4 series and B-1 series；
Wherein, N1～N4 series is with the sea water containing bacterium source for water sample, and in distilled water 1000mL, above-mentioned enrichment medium is: glucose 18～20g, yeast powder 1～1.2g, KH2PO43～4g, NaCl4.5～5g, carbamide 2～3g, pH are 7.2～7.4；
B-1 series is with the oil-polluted water containing bacterium source for water sample, and in distilled water 1000mL, above-mentioned crude oil culture medium is: crude oil 1.5～2g, Na2HPO43～4g, KH2PO43～4g, (NH4)2SO44～5g, NaCl4～5g, MgSO4·7H2O0.5～0.7g, CaCl20.02～0.03g, pH are 7.2～7.4。
Further, heretofore described Rhodopseudomonas is at least one in Pseudomonas aeruginosa Aeruginasa or Pseudomonas rubrisubalbicans Rubrisubalbicans, described pimelobacter sp is simple pimelobacter sp Pimerobactersimplex, described sea Coccus is addicted to salt sea coccus Marinococcushalophilus, described Microbacterium is addicted at least one in ammonia microbacterium ATCC15354MicrobacteriumammoniaphilumATCC15354 or pretty young woman microbacterium Microbacteriumimperiale, described Planococcus is lemon yellow planococcus Planococcuscitreus。
This invention address that the technical scheme that second technical problem adopts is: the preparation method of above-mentioned immobilized microorganism microbial inoculum comprises the following steps: (1) by after cultivating addicted to salt petroleum hydrocarbon degradation thalline bacterium solution centrifugal 4～5min under 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) drying and crushing after mussel shell cleaning, cross 40～80 mesh sieves, joining in the citric acid solution that concentration is 2～5wt% by the mussel shell powder after sieving, left at room temperature 1～2h after stirring, wherein the plastid of mussel shell powder and citric acid solution is than for 1:1～3；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 120～150 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:2～5 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5～6%, injects 3.5～4.0%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 6～18h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107～2 × 108cell/g。
This invention address that the 3rd technical scheme that technical problem adopts is: the application in Ship Building and Repairing sewage disposal of the described immobilized microorganism microbial inoculum, and 100ml treatment sewage adds the immobilized microorganism microbial inoculum of its volume 2～4%。
For making this immobilized microorganism microbial inoculum that the treatment effect of Ship Building and Repairing sewage disposal is better, as preferably, the salinity of pending described Ship Building and Repairing sewage is 10～12.2 ‰。
Further, the treatment temperature of described Ship Building and Repairing sewage is 25～35 DEG C, and pH is 7～7.5。
Compared with prior art, it is an advantage of the current invention that: in the immobilization material of the present invention, the bio-compatibility of sodium alginate is good, nontoxic, easily fix, the mussel shell powder added, this material source is wide, cheap, the immobilization material being added with mussel shell powder is provided that the mechanical strength of immobilized microorganism microbial inoculum, and improves its mass transfer performances, make nutrient mass-energy be easier to be absorbed by thalline, greatly improve the activating agent degradation efficiency of microorganism。In addition, this immobilized microorganism bacterial preparation process is easy, easy controlled operation, the immobilization made is less than being prone to preserve and recycling, to the degradation rate of oil pollutant in Ship Building and Repairing waste water up to 82～94%, with free microbial ratio, its degradation rate improves 39～47%, and the toleration of environment is strengthened。
Accompanying drawing explanation
Fig. 1 is the Gram’s staining photo figure of N1 bacterium in the embodiment of the present invention；
Fig. 2 is the Gram’s staining photo figure of N2 bacterium in the embodiment of the present invention；
Fig. 3 is the Gram’s staining photo figure of N3 bacterium in the embodiment of the present invention；
Fig. 4 is the Gram’s staining photo figure of N4 bacterium in the embodiment of the present invention；
Fig. 5 is the Gram’s staining photo figure of B-1 bacterium in the embodiment of the present invention；
Fig. 6 is the initial pH impact on N1-N4 series bacteria growing in the embodiment of the present invention；
Fig. 7 is the salinity impact on N1-N4 series bacteria growing in the embodiment of the present invention；
Fig. 8 is the temperature impact on N1-N4 series bacteria growing in the embodiment of the present invention；
Fig. 9 is the initial pH impact on B-1 bacteria growing in the embodiment of the present invention；
Figure 10 is the salinity impact on B-1 bacteria growing in the embodiment of the present invention；
Figure 11 is the temperature impact on B-1 bacteria growing in the embodiment of the present invention。
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail。
Embodiment 1: addicted to the acquisition of salt petroleum hydrocarbon degradation thalline
1 addicted to the screening of salt petroleum hydrocarbon degradation bacterium and qualification
(1) strain source: in the present invention addicted to salt petroleum hydrocarbon degradation thalline by Rhodopseudomonas Pseudomonas, pimelobacter sp belong to Pimelobacter, sea Coccus Marinococcus, Microbacterium Microbacterium and Planococcus Planococcus form。In the present embodiment, Rhodopseudomonas is B-1, and antibacterial respectively N1, N2, N3 and N4 of pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus, wherein N1-N4 series derives from sea water (water sampling in the group Lu Jia that sets sail stand erect surrounding waters, Ship Building and Repairing base), and B-1 series derives from oil-polluted water (water sampling in the group Lu Jia that sets sail stand erect Ship Building and Repairing base)。
(2) culture medium adopted is as follows:
N1-N4 series:
Enrichment medium: Carnis Bovis seu Bubali cream 3.00g, peptone 10.00g, NaCl5.00g, distilled water 1000mL, pH are 7.20～7.40；
Crude oil culture medium: crude oil 2.00g, Na2HPO43.00g, KH2PO43.00g, (NH4)2SO45.00g, NaCl5.00g, MgSO4·7H2O0.50g, CaCl20.02g, distilled water 1000mL, pH are 7.20～7.40。
B-1 series:
Enrichment medium: glucose 20.00g, yeast powder 1.00g, KH2PO43.00g, NaCl5.00g, carbamide 3.00g, distilled water 1000mL, pH are 7.20～7.40；
Crude oil culture medium: crude oil 2.00g, Na2HPO43.00g, KH2PO43.00g, (NH4)2SO45.00g, NaCl5.00g, MgSO4·7H2O0.50g, CaCl20.02g, distilled water 1000mL, pH are 7.20～7.40
(3) experimental technique
The antibacterial of screening is carried out Physiology and biochemistry property testing with reference to " the simple and clear outstanding Bacteria Identification handbook of 8th edition uncle ", " common bacteria system identification handbook " by experiment, by antibacterial Preliminary Identification to belonging to。Authentication method mainly has Gram staining method, spore staining method, V-P experiment, Starch Hydrolysis test, methyl red test, catalase test, gelatin liquefaction, semi-solid agar puncture, mobility's observation of antibacterial, hydrogen sulfide reaction etc.。
(3.1) Gram’s staining: a small amount of lawn of picking is coated on microscope slide, air-dry, fixes smear on flame, drips violet staining 1～2min, washing, and dropping iodine liquid washes away back water, and covers about 1min with iodine liquid, washing。Suck the back water on microscope slide with filter paper, slide is tilted, under white background, with the ethanol decolorization of 95%, until when the ethanol flowed out is without purple, washing immediately。About 2min is redyed, washing with sarranine liquid。After drying, with oil sem observation。Thalline is dyed to hepatic for gram positive bacteria, be dyed to redness for gram negative bacteria。
(3.2) spore staining: add 1～2 drip in small test tube, with inoculating loop picking 2～3 ring lawn in test tube, stir, make dense bacteria suspension。Add peacock green dye 2～3 in small test tube, and make it mix homogeneously with bacterium solution, then test tube is placed in the beaker of boiling water bath, heat dyeing 15～20min。In clean glass slide, thin film is painted with bacterium solution ring of numbers bottom inoculating loop picking test tube, then that smear is fixing by 3 warms of flame。Washing, till the water redgreen flowed out。Dyeing 2～3min with sarranine dye liquor, incline dye liquor blot residual liquid with filter paper。Dried with oil sem observation。Spore is in green, and sporangium and trophosome are red。
(3.3) catalase experiment: by the 24h inclined plane inoculating cultivated, pick with Glass rod on the slide being coated in 3% hydrogen peroxide a little, produces to be then positive, bubble-free is negative if any bubble。
(3.4) C.I. 13020. experiment: culture medium: peptone, 5g；Glucose, 5g；K2HPO4, 5g。Adding a C.I. 13020. reagent in culture fluid, red is methyl red test positive reaction, and yellow is negative reaction。
(3.5) V-P test: the same methyl red test of culture medium。Take culture fluid and 40% sodium hydroxide equivalent is mixed mutually。Adding a little creatine, there is redness in 10min such as culture fluid, is test positive reaction。
(3.6) Starch Hydrolysis: add 0.2% soluble starch in gravy peptone, make flat board。Cultivating 2-5 days, after forming obvious bacterium colony, it is black-and-blue for dripping iodine liquid flat board on flat board, and periphery of bacterial colonies, if any invariant color transparent circle, represents that Starch Hydrolysis is positive, is still black-and-blue for feminine gender。
(3.7) cellulose hydrolysis: culture medium: peptone, 5g；NaCl, 5g。By culture medium subpackage test tube, in the medium soak a high-quality filter paper, filter paper bar can be resolved into a fiber paper slip is fractureed or thinning person for the positive, unchanged person be feminine gender。
(3.8) gelatin liquefaction: culture medium: peptone, 5g；Gelatin, 100-150g。Subpackage test tube, takes the slant culture percutaneous puncture-inoculation of 18-24h, does blank, cultivates in 20 DEG C of incubators, and room temperature less than 20 DEG C observes whether gelatin liquefies, and as bacterium grows, gelatin surface is without depression and be stable grumeleuse, then be gelatin hydrolysis feminine gender。Such as gelatin clot parts or be all flowable liquid below 20 DEG C, then it it is gelatin liquefaction positive。
(4) bacterial screening result
Above-mentioned water sample is respectively connected to enrichment medium 25 DEG C activation, then will inject after the bacterium solution after activation in oil culture medium, culture bottle is placed in shaking table 25 DEG C, 180r/min cultivates domestication, finally with oil flat board carry out repeatedly separate, purification obtain N1-N4 series (Fig. 1, Fig. 2, Fig. 3 and Fig. 4), B-1 series (Fig. 5)。
Wherein B-1 bacterium is surfactant producing bacteria, by filtration, bactofugation body after B-1 in the medium fermentation culture, takes certain supernatant and adjusts pH=2, place refrigerator overnight, without precipitation generation, so B-1 bacterium biosurfactant production is initially identified as glycolipid class。
(5) qualification result of strain
N1, N2, N3, N4 bacterium is carried out the observation of bacterium colony configuration of surface and the mensuration of Physiology and biochemistry character, by above-mentioned 5 bacterial strain Preliminary Identification to belonging to, wherein B-1 is Rhodopseudomonas, N1 is that pimelobacter sp belongs to, N2 is sea Coccus, N3 is Microbacterium, and N4 is Planococcus, and surface morphology characteristics and the physiological and biochemical property of various bacterium are as described in Table 1。
The surface morphology characteristics of table 1 thalline and physiological and biochemical property
2 microbial inoculum exploitations
(1) culture medium used in microbial inoculum exploitation:
NI-N4 series:
First enrichment medium: Carnis Bovis seu Bubali cream 3.00g, peptone 10.00g, NaCl5.00g, distilled water 1000ml, pH are 7.20-7.40。
B-1 series:
Second enrichment medium: glucose 20.00g, yeast powder 1.00g, KH2PO43.00g, NaCl5.00g, carbamide 3.00g, distilled water 1000ml, pH are 7.20-7.40。
(2) experimental technique
The evaluation of strain is respectively with culture medium OD600Value or surface tension decreasing value represent the situation that strain grows at different conditions。
(3) impact on oil degradation bacteria NI-N4 series and B-1 growth of pH, salinity and temperature is investigated respectively。
(3.1) NI-N4 series
(3.1.1) initial pH is on the surfactant hydrocarbon degradation bacteria N1-N4 impact grown
Regulate the pH respectively 5,6,7,8 and 9 into enrichment medium, in being connect with identical inoculum concentration by 4 bacterial strains, 25 DEG C, after 180r/min cultivates 3d, measure the OD of culture fluid600Value。As can be seen from Figure 6,4 bacterial strains are well-grown in neutral slight alkali environment, and owing to the pH of actual sea water is 7.6～8.5, coastal vessel is built enterprise and adopted sea water to be carried out waiting operation, the general meta-alkalescence of the pH containing sea water waste water produced, the bacterial strain therefore screened may be used for actual improvement。
(3.1.2) salinity is on the surfactant hydrocarbon degradation bacteria N1-N4 impact grown
By 4 bacterial strains with identical inoculum concentration access the mass fraction of NaCl respectively 1%, 3%, 5%, 7% and 9% enrichment medium in, 25 DEG C, 180r/min cultivates 3d, the OD of mensuration culture fluid600Value。It can be seen from figure 7 that 4 bacterial strains well-grown when salinity (i.e. the mass fraction of NaCl) is 3%, being suitable for the saline environment of Ship Building and Repairing waste water, and N1 and N4 has good salt tolerance, therefore bacterial strain can meet actual operation requirements completely。
(3.1.3) temperature is on the surfactant hydrocarbon degradation bacteria N1-N4 impact grown
By in 4 bacterial strains enrichment medium with identical inoculum concentration cut-in temperature respectively 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C and 35 DEG C, 180r/min cultivates 3d, measures the OD of culture fluid600Value。From figure 8, it is seen that this 4 bacterial strain well-grown when 20-30 DEG C, and all there is good cold resistance。
(3.2) B-1 bacterium
(3.2.1) initial pH is on the B-1 impact grown
250mL triangular flask loads 100mL enrichment medium, regulates the pH respectively 5,6,7,8,9,10 of culture fluid, B-1 bacterium is accessed in enrichment medium, 25 DEG C, after 180r/min cultivates 3d, measure the surface tension value of culture fluid。B-1 grows fine within the scope of pH7-9 as seen from Figure 9, produces Surfactant amount more。
(3.2.2) salinity is on the B-1 impact grown
By 4 bacterial strains with identical inoculum concentration access the mass fraction of NaCl respectively 1%, 3%, 5%, 7% and 9% enrichment medium in, 25 DEG C, 180r/min cultivates 3d, the OD of mensuration culture fluid600Value。B-1 is well-grown within the scope of 3-5 in salinity as seen from Figure 10, has stronger salt tolerance。
(3.2.3) temperature is on the B-1 impact grown
By in the B-1 bacterium enrichment medium with identical inoculum concentration cut-in temperature respectively 5 DEG C, 15 DEG C, 25 DEG C and 35 DEG C, 180r/min cultivates 3d, measures the OD of culture fluid600Value。It can be seen from figure 11 that bacterial strain well-grown when 15-35 DEG C, and there is good cold resistance。
The immobilized microorganism thalline Degrading experiment to oil-polluted water:
Being 2% be inoculated in immobilized microbial inoculum microsphere in the sterilized 100mL oil-polluted water prepared according to volume ratio, be placed in constant temperature oscillation shaking table 25 DEG C of degradeds, shaking speed is 150r/min。
The mensuration of (a) oil degradation efficiency
The mensuration of crude oil in samples content carries out according to national standard method spectrophotography SY/T0530-2011, measures wavelength and selects 420nm, and is calculated as follows the degradation efficiency (η) of crude oil:
In formula:
C0The concentration (mg/L) of blank oil-polluted water Crude Oil；
C1The concentration (mg/L) of oil-polluted water Crude Oil after degraded。
The mensuration of (b) microbial inoculum Biomass
During sample determination, the microsphere in oil-polluted water is taken out after cleaning, first weigh up the quality of microsphere, then to after its broken addition 25mL sterilized water vibration, take 1mL supernatant blood counting chamber microscopy and measure Biomass, be expressed as cell/g (microsphere)。
Belonged to Pimelobacter addicted to salt petroleum hydrocarbon degradation thalline by Rhodopseudomonas Pseudomonas, pimelobacter sp, sea Coccus Marinococcus, Microbacterium Microbacterium and Planococcus Planococcus forms, and mass ratio is 3～5:1～3:2～3:1～2:1～2。Utilize the N1～N4 series bacterial strain and the B-1 series bacterial strain that obtain in embodiment 1 or adopt commercially available bacterial strain above-mentioned addicted to salt petroleum hydrocarbon degradation thalline to constitute, and then preparation is for the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal, the immobilization material of this immobilized microorganism microbial inoculum includes sodium alginate, CaCl2, mussel shell powder and concentration be the citric acid of 2～5wt%。The preparation process of 2～6 pairs of these immobilized microorganism microbial inoculums is described in detail by the following examples。
Embodiment 2: for the preparation of the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal
In the present embodiment, the mass ratio of mussel shell powder and citric acid is 1:1, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8:0.7。It is B-1 addicted to Rhodopseudomonas in salt petroleum hydrocarbon degradation thalline, pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus respectively N1, N2, N3 and N4, and the mass ratio of B-1, N1, N2, N3 and N4 is 3:1:2:1:1, preparation process comprises the following steps:
(1) by after cultivating addicted to 4min centrifugal under salt petroleum hydrocarbon degradation thalline bacterium solution 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) mussel shell clean after drying and crushing, cross 40 mesh sieves, the mussel shell powder after sieving joined in the citric acid solution that concentration is 5wt%, left at room temperature 2h after stirring, wherein the plastid of mussel shell powder and citric acid solution ratio is for 1:1；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 120 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:2 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5%, injects 3.5%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 6h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107cell/g。
Embodiment 3: for the preparation of the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal
In the present embodiment, the mass ratio of mussel shell powder and citric acid is 1:2, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:1:0.8。It is B-1 addicted to Rhodopseudomonas in salt petroleum hydrocarbon degradation thalline, pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus respectively N1, N2, N3 and N4, and the mass ratio of B-1, N1, N2, N3 and N4 is 4:3:3:2:2, preparation process comprises the following steps:
(1) by after cultivating addicted to 5min centrifugal under salt petroleum hydrocarbon degradation thalline bacterium solution 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) mussel shell clean after drying and crushing, cross 80 mesh sieves, the mussel shell powder after sieving joined in the citric acid solution that concentration is 3wt%, left at room temperature 1h after stirring, wherein the plastid of mussel shell powder and citric acid solution ratio is for 1:2；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 150 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:3 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 6%, injects 4.0%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 12h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 2 × 108cell/g。
Embodiment 4: for the preparation of the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal
In the present embodiment, the mass ratio of mussel shell powder and citric acid is 1:3, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8:0.7, it is B-1 addicted to Rhodopseudomonas in salt petroleum hydrocarbon degradation thalline, pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus respectively N1, N2, N3 and N4, and the mass ratio of B-1, N1, N2, N3 and N4 is 5:2:2:1:2, preparation process comprises the following steps:
(1) by after cultivating addicted to 5min centrifugal under salt petroleum hydrocarbon degradation thalline bacterium solution 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) mussel shell clean after drying and crushing, cross 60 mesh sieves, the mussel shell powder after sieving joined in the citric acid solution that concentration is 2～5wt%, left at room temperature 1.5h after stirring, wherein the plastid of mussel shell powder and citric acid solution ratio is for 1:3；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 130 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:5 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5%, injects 4.0%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 18h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 7 × 107cell/g。
Embodiment 5: for the preparation of the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal
In the present embodiment, the mass ratio of mussel shell powder and citric acid is 1:1, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8:0.7。It is Pseudomonas aeruginosa Aeruginasa addicted to Rhodopseudomonas in salt petroleum hydrocarbon degradation thalline, pimelobacter sp belongs to, sea Coccus, Microbacterium and Planococcus antibacterial respectively simple pimelobacter sp Pimerobactersimplex, addicted to salt sea coccus Marinococcushalophilus, addicted to ammonia microbacterium ATCC15354MicrobacteriumammoniaphilumATCC15354 and lemon yellow planococcus Planococcuscitreus, all it is purchased from Shanghai richness many (sub-Pingning) biotechnology Development Co., Ltd, and mass ratio is 5:1:2:2:2。Preparation process comprises the following steps:
(1) by after cultivating addicted to 4min centrifugal under salt petroleum hydrocarbon degradation thalline bacterium solution 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) mussel shell clean after drying and crushing, cross 40 mesh sieves, the mussel shell powder after sieving joined in the citric acid solution that concentration is 5wt%, left at room temperature 2h after stirring, wherein the plastid of mussel shell powder and citric acid solution ratio is for 1:1；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 120 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:2 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5%, injects 3.5%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 6h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 1 × 108cell/g。
Embodiment 6:
Preparation for the immobilized microorganism microbial inoculum of Ship Building and Repairing sewage disposal
In the present embodiment, the mass ratio of mussel shell powder and citric acid is 1:1, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8:0.7。It is Pseudomonas rubrisubalbicans Rubrisubalbicans addicted to Rhodopseudomonas in salt petroleum hydrocarbon degradation thalline, pimelobacter sp belongs to, sea Coccus, Microbacterium and Planococcus antibacterial respectively simple pimelobacter sp Pimerobactersimplex, addicted to salt sea coccus Marinococcushalophilus, pretty young woman microbacterium Microbacteriumimperiale and lemon yellow planococcus Planococcuscitreus, all it is purchased from Shanghai richness many (sub-Pingning) biotechnology Development Co., Ltd, and mass ratio is 3:3:3:2:2。Preparation process comprises the following steps:
(1) by after cultivating addicted to 4min centrifugal under salt petroleum hydrocarbon degradation thalline bacterium solution 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) mussel shell clean after drying and crushing, cross 40 mesh sieves, the mussel shell powder after sieving joined in the citric acid solution that concentration is 5wt%, left at room temperature 2h after stirring, wherein the plastid of mussel shell powder and citric acid solution ratio is for 1:1；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 120 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:2 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5%, injects 3.5%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 6h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107cell/g。
Embodiment 7: the immobilized microorganism thalline cleaning test to Ship Building and Repairing waste water
Appoint the immobilized microorganism microbial inoculum of preparation in Example 2～embodiment 7, analyze it to the removal effect to Ship Building and Repairing waste water Petroleum Hydrocarbon pollutant etc., and compare with free microorganism microbial inoculum。
Waste water used by the present embodiment is taken from the group Lu Jia that sets sail and is stood erect Ship Building and Repairing base sewage collecting basin, and water quality index is as follows: COD, 460 ± 21.6mg/L；BOD5,220.0 ± 23.5mg/L；Petroleum-type: 60.4 ± 5.8mg/L；NH4+-N, 25.3 ± 9.0mg/L；TN, 30.2 ± 7.2mg/L；TP, 2.6 ± 0.9mg/L；SS, 157.0 ± 40.8mg/L, salinity is 12.2 ‰。
Result of the test is as shown in table 2, is 12.2 ‰ in salinity, and temperature is 25 DEG C, pH be 7.5 environment in the degradation rate of oil pollutant in Ship Building and Repairing waste water up to 82～94%, with free microbial ratio, its degradation rate improves 39～47%, and environmental resistance is strengthened。
As can be seen from Table 2, the immobilized microorganism microsphere adding modified mussel shell improves a lot in mechanical strength and mass-transfer performance, the cell of embedding has higher activity, to the degradation rate of Ship Building and Repairing waste water Petroleum Hydrocarbon up to 94%, the degradation rate of COD is reached 95%, obviously higher than being not added with material and with the addition of the immobilized microorganism microsphere of inorganic active carbon。Modified mussel shell is added in sodium alginate-calcium chloride entrapped immobilized carrier, improve the mechanical strength of preparation, and due to the loose structure of modified mussel shell, it is greatly improved its mass transfer performances, nutrient substance is made to be easier to, by microorganism panning, drastically increase the activating agent degradation efficiency of microorganism。Modified mussel shell is compared with general adsorbent-activated carbon, and raw material sources are more extensive, and price is more cheap, and makes the performance of immobilized microorganism more excellent, and discarded mussel shell recycling, solves environmental problem and also achieves the resource of discarded mussel shell。
The different immobilized microorganism microsphere Performance comparision in Ship Building and Repairing wastewater treatment of table 2
Claims (9)
1. the immobilized microorganism microbial inoculum for Ship Building and Repairing sewage disposal, it is characterised in that including addicted to salt petroleum hydrocarbon degradation thalline and immobilization material, this immobilization material includes sodium alginate, CaCl2, mussel shell powder and concentration be the citric acid of 2～5wt%, wherein mussel shell powder is 1:1～3 with the mass ratio of citric acid, mussel shell powder and sodium alginate and CaCl2Mass ratio be 1:0.8～1:0.7～0.8。
2. immobilized microorganism microbial inoculum as claimed in claim 1, it is characterised in that described immobilized microorganism microbial inoculum is microspheroidal, and wherein the concentration addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107～2 × 108cell/g。
3. immobilized microorganism microbial inoculum as claimed in claim 2, it is characterized in that, described addicted to salt petroleum hydrocarbon degradation thalline by Rhodopseudomonas Pseudomonas, pimelobacter sp belong to Pimelobacter, sea Coccus Marinococcus, Microbacterium Microbacterium and Planococcus Planococcus form, mass ratio is 3～5:1～3:2～3:1～2:1～2。
4. immobilized microorganism microbial inoculum as claimed in claim 3, it is characterized in that, described Rhodopseudomonas is B-1, antibacterial respectively N1, N2, N3 and N4 of described pimelobacter sp genus, sea Coccus, Microbacterium and Planococcus, and obtain each through following steps: 25 DEG C of activation in enrichment medium will be accessed containing bacterium source water sample, bacterium solution after activation is injected in crude oil culture medium, 25 DEG C, 180～200r/min cultivate domestication, finally with oil flat board carry out repeatedly separate, purification obtain N1～N4 series and B-1 series；
Wherein, N1～N4 series, with the sea water containing bacterium source for water sample, in the enrichment medium of distilled water 1L, above-mentioned N1～N4 series is: Carnis Bovis seu Bubali cream 3～4g, peptone 9～10g, NaCl4.5～5g, pH are 7.2～7.4；
In the crude oil culture medium of distilled water 1L, above-mentioned N1～N4 series it is: crude oil 1.5～2g, Na2HPO43～4g, KH2PO43～4g, (NH4)2SO44～5g, NaCl4～5g, MgSO4·7H2O0.5～0.7g, CaCl20.02～0.03g, pH are 7.2～7.4；
B-1 series, with the oil-polluted water containing bacterium source for water sample, in the enrichment medium of distilled water 1L, above-mentioned B-1 series is: glucose 18～20g, yeast powder 1～1.2g, KH2PO43～4g, NaCl4.5～5g, carbamide 2～3g, pH are 7.2～7.4；
In the crude oil culture medium of distilled water 1L, above-mentioned B-1 series it is: crude oil 1.5～2g, Na2HPO43～4g, KH2PO43～4g, (NH4)2SO44～5g, NaCl4～5g, MgSO4·7H2O0.5～0.7g, CaCl20.02～0.03g, pH are 7.2～7.4。
5. immobilized microorganism microbial inoculum as claimed in claim 3, it is characterized in that, described Rhodopseudomonas is at least one in Pseudomonas aeruginosa Aeruginasa or Pseudomonas rubrisubalbicans Rubrisubalbicans, described pimelobacter sp is simple pimelobacter sp Pimerobactersimplex, described sea Coccus is addicted to salt sea coccus Marinococcushalophilus, described Microbacterium is addicted at least one in ammonia microbacterium ATCC15354MicrobacteriumammoniaphilumATCC15354 or pretty young woman microbacterium Microbacteriumimperiale, described Planococcus is lemon yellow planococcus Planococcuscitreus。
6. the preparation method of the immobilized microorganism microbial inoculum in Claims 1 to 5 as described in any claim, it is characterised in that comprise the following steps:
(1) by after cultivating addicted to salt petroleum hydrocarbon degradation thalline bacterium solution centrifugal 4～5min under 3000rpm, remove the supernatant, collect bottom concentration bacterium solution, regulated to OD with sterilized water600It is 1, obtains addicted to salt petroleum hydrocarbon degradation thalline concentrated solution；
(2) drying and crushing after mussel shell cleaning, cross 40～80 mesh sieves, joining in the citric acid solution that concentration is 2～5wt% by the mussel shell powder after sieving, left at room temperature 1～2h after stirring, wherein the plastid of mussel shell powder and citric acid solution is than for 1:1～3；
(3) above-mentioned mixed solution is put in Muffle furnace activate, take out cooling after activation, and the sample after activation is pulverized again or crosses 120～150 mesh sieves, obtain modified mussel shell powder；
(4) by this modified mussel shell powder with carry out mixing and absorption addicted to salt petroleum hydrocarbon degradation thalline concentrated solution 1:2～5 in mass ratio, when adsorb saturated time, the modified mussel shell powder of antibacterial must be adsorbed；
(5) adding concentration in the modified mussel shell powder of above-mentioned absorption antibacterial is the sodium alginate soln of 5～6%, injects 3.5～4.0%CaCl with balling-up equipment after mixing2Disperseing balling-up in solution, cross-link 6～18h, obtain immobilized microorganism microbial inoculum, in this immobilized microorganism microbial inoculum, the content addicted to salt petroleum hydrocarbon degradation thalline is 5 × 107～2 × 108cell/g。
7. the application of the immobilized microorganism microbial inoculum in Claims 1 to 5 as described in any claim, it is characterized in that, the application in Ship Building and Repairing sewage disposal of the described immobilized microorganism microbial inoculum, and 100ml treatment sewage adds the immobilized microorganism microbial inoculum of its volume 2～4%。
8. apply as claimed in claim 7, it is characterised in that the salinity of described Ship Building and Repairing sewage is 10～12.2 ‰。
9. applying as claimed in claim 7, it is characterised in that the treatment temperature of described Ship Building and Repairing sewage is 25～35 DEG C, pH is 7～7.5。
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