CN104830721A - Bacterium AJ07 having petroleum degradation function, use thereof, and seabed settlement petroleum degradation bacterium agent - Google Patents
Bacterium AJ07 having petroleum degradation function, use thereof, and seabed settlement petroleum degradation bacterium agent Download PDFInfo
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- CN104830721A CN104830721A CN201510218059.2A CN201510218059A CN104830721A CN 104830721 A CN104830721 A CN 104830721A CN 201510218059 A CN201510218059 A CN 201510218059A CN 104830721 A CN104830721 A CN 104830721A
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- 230000015556 catabolic process Effects 0.000 title claims abstract description 82
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 82
- 239000003208 petroleum Substances 0.000 title claims abstract description 33
- 241000894006 Bacteria Species 0.000 title claims abstract description 23
- 239000003795 chemical substances by application Substances 0.000 title abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 53
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
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- 230000001580 bacterial effect Effects 0.000 claims description 35
- 239000002068 microbial inoculum Substances 0.000 claims description 34
- 238000004062 sedimentation Methods 0.000 claims description 24
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- 239000007788 liquid Substances 0.000 claims description 12
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims description 10
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- 239000004021 humic acid Substances 0.000 claims description 10
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
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- 102000016938 Catalase Human genes 0.000 claims description 2
- 108010053835 Catalase Proteins 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- 241000345369 Edwardsiella hoshinae Species 0.000 claims description 2
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- 108090000913 Nitrate Reductases Proteins 0.000 claims description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
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- 239000008107 starch Substances 0.000 claims description 2
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- 241001425152 Rhodococcus aetherivorans Species 0.000 description 3
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- 241001156739 Actinobacteria <phylum> Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- 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
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
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- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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Abstract
The invention relates to a bacterium AJ07 having a petroleum degradation function, a use thereof, and a seabed settlement petroleum degradation bacterium agent, and belongs to the technical filed of microbes. The bacterium AJ07 is used for petroleum pollution treatment in a petroleum degradation mode. The use of the bacterium is realized in a form of settlement bacterium agent particles. The bacterium agent particles are obtained through the following steps: activating a culture material, and preparing a breeding medium; carrying out strain culture enlargement to prepare a fermentation material of strain suitable for petroleum degradation; and carrying out mixing granulation. The bacterium AJ07 suitable for petroleum degradation is screened from marine indigenous microorganisms for the first time. The seabed settlement petroleum degradation bacterium agent developed in the invention makes petroleum degradation microorganisms have living space and obtain slow release nutrition. The degradation bacterium agent has the settlement characteristic, and solves the problems of seabed settlement petroleum pollution induced by petroleum pollution, and seabed desertification initiated by seabed settlement petroleum pollution.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a strain and there is the strains A J07 of oil degradation function and utilize the application in this bacterial strain Marine Environmental Governance field, the application in the biological treating field of the petroleum pollution caused after especially relating to petroleum pollution.
Background technology
In recent years along with the transport of marine oil, exploitation, handling, processing and use, marine oil incident of leakage happens occasionally, and petroleum pollution in ocean is increasingly sharpened.Oil is serious to the destruction of species diversity, and comprise the common various fish of people, birds and other biology and all receive and have a strong impact on, oil can attach on the fish gill, fish is suffocated, suppress aquatic bird lay eggs and hatch, destroy the watertightness of its feather, reduce aquatic product quality; Meanwhile, the oil of leakage can form oil film, hinders the reoxygenation effect of water body, affects marine plankton growth, destroys marine ecology balance; Can beachscape be destroyed in addition, affect beach aesthetic values.
At present; be directed to petroleum pollution; especially big area Oil spills; the emergency schedule of usual employing is except the physical methods such as artificial salvaging that recover the oil; also adopt and add oil spill dispersant and make petroleum emulsification, the method for rapid sedimentation is to recover temporary transient the cleaning, although this method seems the effect serving improvement in short-term in sea; but under cover crisis behind, it can cause secondary chemical pollution.
Therefore, the reconstruction that enforcement physico-chemical process carries out ecotope may realize hardly, and biological restoration pollution of the sea becomes the approach of unique feasible.But relative to land, marine plant is relatively deficient, the biological restoration of Marine Pollution also mainly relies on the Degradation of marine microorganism, especially depends on the various indigenous oil degradation microorganism having adapted to environments such as subsea.These oil degradation microorganisms can utilize oil as its sole carbon source in seabed, overcome the extreme condition of seabed dark, low temperature, oligotrophy, anoxic, take oil as food, oil is carried out decomposition and inversion, thus change and aqueous desert problem.
Summary of the invention
The pollution problem caused is revealed, as the pollution problem of the floating oil on sea in order to solve oil; The and for example secondary pollution of chemical harnessing initiation, especially in order to the seabed sedimentation petroleum pollution caused after solving petroleum pollution and the aqueous desert problem then caused, contriver reaches out for efficient ocean original inhabitants' petroleum microorganism and studies, and finally obtains the strains A J07 that a strain is applicable to oil degradation.Contriver requires to protect its purposes while this bacterial strain claimed.
Technical scheme of the present invention is as follows:
One strain has the strains A J07 of oil degradation function, Latin called after Rhodococcusaetherivorans.Its 16SrRNA sequence is as shown in SEQ ID No.1.
Described strains A J07 is that contriver is separated the strain food ether rhodococcus obtained from occurring in nature.Strains A J07 belongs to actinomycetes door (Actinobacteria), Actinomycetes (Actinobacteria), actinomycetes subclass (Actinobacteridae), actinomycetales (Actinobacterales), bar bacterium suborder (Corynebacterineae), Nocardiaceae (Nocardiaceae), Rhod (Rhodococcus).
The direct sources of strains A J07 is that contriver is separated and obtains from the marine bottom sediment that Talien New Port petroleum pollution marine site gathers.Get 5 grams, mud sample sterilized water dilution 10
5doubly, by crude oil even spread on ASM substratum, cultivate separation in 7 days for 15 DEG C and obtain bacterial strain.Strains A J07 is separated and obtains from the marine bottom sediment that Talien New Port petroleum pollution marine site gathers, and picker's name Liu length is built, picker's contact method 0411-87656046.
The described strains A J07 being applicable to oil degradation submits preservation to, and concrete preservation information is as follows:
Depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);
Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica;
Preservation date: on 01 13rd, 2015;
Deposit number: CGMCC No.10350;
The Morphology and physiology biochemical character of described strains A J07 is:
(1) colony morphology characteristic
LB substratum was cultivated after 7 days, and in orange, rat is opaque, slightly gloss, smooth, and colony edge is neatly smooth.
(2) strain morphology and physio-biochemical characteristics thereof
Gram positive aerobic bacterium, catalase and nitrate reductase enzyme positive, edwardsiella hoshinae, not hydrolyzed starch, thalline rod-short, 1.1-1.6*0.5-0.7 μm.All can grow within the scope of NaCl concentration 0-20%, the suitableeest NaCl concentration 3%, the temperature range 10-50 DEG C of growth, optimum temperature range 28-37 DEG C, pH scope 5-10.5, optimal pH 7.5-8.5, sucrose, fructose and lactose can not be utilized, glucose and N.F,USP MANNITOL can be utilized.
The purposes of described strains A J07 is carry out the improvement of petroleum pollution in the mode of oil degradation.
Preferably, this purposes be with can sedimentation microbial inoculum particle form realize.
First technical conceive of contriver is: research and develop a kind of oil degradation microorganism carrier, can not only provide the space lived away from home for strains A J07, and this carrier can be strains A J07 simultaneously provides slow-release nutrient and possesses settling character.
This object of the present invention is realized by following proposal:
Seabed sedimentation oil degradation microbial inoculum is by the mass ratio of the raw materials of the fermentation material of bacterial classification AJ07 and oil degradation microbe carrier according to 1:5-15, and after the mass ratio mixing of preferred 1:10, granulation is dried and obtains; Further, final bacteria suspension concentration is made to be 10
10more than cfu/ml is good;
Raw materials and the proportioning thereof of described oil degradation microbe carrier comprise:
| Humic acid | 50-80wt％ |
| Groundnut meal | 2-5wt％ |
| Wheat bran | 2-5wt％ |
| Wilkinite | 15-39wt％ |
| Na 2HPO 4 | 0.5-0.7wt％ |
| KH 2PO 4 | 0.1-0.2wt％ |
| NH 4NO 3 | 0.2-0.3wt％ |
。
Further, the fermentation material of described bacterial classification AJ07 can adopt any one fermentation process known to ordinary skill in the art to obtain.But the following methods adopting contriver to use produces fermentation material, can obtain better effect:
The fermentation material of bacterial classification AJ07 is by the bacterial classification AJ07 culture material that activated and propagating culture medium according to 1:5-10 mass ratio, the mass ratio mixing of preferred 1:10; Solid fermentation 3 ~ 7 days at 20 ~ 50 DEG C, preferably 37 DEG C, preferably fermentation 5 days, obtains the fermentation material of bacterial classification AJ07, further, makes the viable count 10 of final fermentation material
10more than cfu/g;
Described activation good bacterial classification AJ07 culture material be to access in activated liquid LB substratum at 25-50 DEG C at the LB inclined-plane picking strains A J07 being suitable for oil degradation to be activated, preferably 37 DEG C carry out activating and obtain; Thus to obtain activating rear cell concentration be 10
10cfu/ml culture material; Described activated liquid LB substratum is: peptone 10g/L, NaCl 10g/L, yeast extract paste 5g/L, pH 7.0 ~ 7.5; 121 DEG C, 30min sterilizing;
Described propagating culture medium solid feed and proportioning:
| Humic acid | 50-80wt％ |
| Groundnut meal | 2-5wt％ |
| Wheat bran | 3-8wt％ |
| Wilkinite | 15-37wt％ |
The humidity of propagating culture medium finished product preferably 55%.
Further, described granulation is dried can conventionally granulation, but the method for preferred following contriver's employing, more excellent effect can be produced:
It is added by compound in stirrer and add binding agent to stir that granulation is dried, and the fully mixing that adds water makes water content lower than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents; Namely obtain seabed sedimentation oil degradation microbial inoculum after cooling, ensure that in seabed sedimentation oil degradation microbial inoculum, viable count reaches 10
9cfu/ml;
Preferably, described following current is dried, and specifically enters in dram by wet granular by the intermediate bin of dryer front portion, meanwhile hot blast enters dram in a co-current manner, under in rotary barrel, flight drives, material is taken up, and forms point material curtain spreading uniform comparatively dense;
Preferably, the raw materials of oil degradation microbe carrier is the fine powder of below 20 orders;
Preferably, the raw materials of propagating culture medium is the fine powder of below 20 orders.
Beneficial effect of the present invention is:
1. the present invention filters out the strains A J07 being applicable to oil degradation first from the indigenous microorganism of ocean;
2. a kind of submarine oil degradation bacterial agent of contriver's research and development, not only makes oil degradation microorganism have the space lived away from home, and microorganism can obtain the nutrition of slowly-releasing simultaneously; This degradation bacterial agent possesses settling character, the seabed sedimentation petroleum pollution caused after solving petroleum pollution and the aqueous desert problem then caused.
Accompanying drawing explanation
The colonial morphology figure of Fig. 1 strains A J07;
The sem observation figure of the thalline of Fig. 2 strains A J07;
The phylogenetic tree of Fig. 3 strains A J07;
The oil degradation performance measurement of Fig. 4 strains A J07;
In the drawings: A is for adding oil degradation bacterial strain AJ07 treatment effect figure, and B contrasts design sketch for not adding oil degradation bacterial strain AJ07;
Fig. 5 is the trophism Parameter Map of microbial inoculum carrier to strains A J07;
Fig. 6 strains A J07 microbial inoculum of the present invention particle form figure.
Embodiment
In the mode specifically implemented, technical scheme of the present invention is further described below, but the present invention is not limited to embodiment content in any form.
One, embodiment
Embodiment 1
Seabed sedimentation oil degradation microbial inoculum is that after being mixed according to the mass ratio of 1:15 with the raw materials of oil degradation microbe carrier by the fermentation material of bacterial classification AJ07, granulation is dried and obtains; Further, final bacteria suspension concentration is made to be 10
10more than cfu/ml is good;
Raw materials and the proportioning thereof of described oil degradation microbe carrier comprise:
| Humic acid | 80wt％ |
| Groundnut meal | 2wt％ |
| Wheat bran | 2wt％ |
| Wilkinite | 15wt％ |
| Na 2HPO 4 | 0.5wt％ |
| KH 2PO 4 | 0.2wt％ |
| NH 4NO 3 | 0.3wt％ |
。
Further, the fermentation material of described bacterial classification AJ07 can adopt any one fermentation process known to ordinary skill in the art to obtain.But the following methods adopting contriver to use produces fermentation material, can obtain better effect:
The fermentation material of bacterial classification AJ07 is that at 50 DEG C, solid fermentation 7 days, obtains the fermentation material of bacterial classification AJ07, further, makes the viable count 10 of final fermentation material by the bacterial classification AJ07 that activated and propagating culture medium according to 1:10 mass ratio
10more than cfu/g;
Described bacterial classification AJ07 culture material be to access in activated liquid LB substratum at 50 DEG C at LB inclined-plane picking strains A J07 to be activated, preferably 37 DEG C carry out activating and obtain; Thus to obtain activating rear cell concentration be 10
10the culture material of cfu/ml; Described activated liquid LB substratum is: peptone 10g/L, NaCl 10g/L, yeast extract paste 5g/L, pH 7.0 ~ 7.5; 121 DEG C, 30min sterilizing;
Described propagating culture medium solid feed and proportioning:
| Humic acid | 80wt％ |
| Groundnut meal | 2wt％ |
| Wheat bran | 3wt％ |
| Wilkinite | 15wt％ |
The humidity of propagating culture medium finished product preferably 55%.
Further, described granulation is dried can conventionally granulation, but the method for preferred following contriver's employing, more excellent effect can be produced:
It is added by compound in stirrer and add binding agent to stir that granulation is dried, and the fully mixing that adds water makes water content lower than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents; Namely obtain seabed sedimentation oil degradation microbial inoculum after cooling, ensure that in seabed sedimentation oil degradation microbial inoculum, viable count reaches 10
9cfu/ml;
Preferably, described following current is dried, and specifically enters in dram by wet granular by the intermediate bin of dryer front portion, meanwhile hot blast enters dram in a co-current manner, under in rotary barrel, flight drives, material is taken up, and forms point material curtain spreading uniform comparatively dense;
Preferably, the raw materials of oil degradation microbe carrier is the fine powder of below 20 orders;
Preferably, the raw materials of propagating culture medium is the fine powder of below 20 orders.
Described dryer can select any one can complete the commercially available dryer of baking step, but the dryer of this model of preferred following manufacturer production: Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
Embodiment 2
Seabed sedimentation oil degradation microbial inoculum is that after being mixed according to the mass ratio of 1:5 with the raw materials of oil degradation microbe carrier by the fermentation material of bacterial classification AJ07, granulation is dried and obtains; Further, final bacteria suspension concentration is made to be 10
10more than cfu/ml is good;
Raw materials and the proportioning thereof of described oil degradation microbe carrier comprise:
| Humic acid | 53.1wt％ |
| Groundnut meal | 5wt％ |
| Wheat bran | 2wt％ |
| Wilkinite | 39wt％ |
| Na 2HPO 4 | 0.5wt％ |
| KH 2PO 4 | 0.2wt％ |
| NH 4NO 3 | 0.2wt％ |
。
Further, the fermentation material of described bacterial classification AJ07 can adopt any one fermentation process known to ordinary skill in the art to obtain.But the following methods adopting contriver to use produces fermentation material, can obtain better effect:
The fermentation material of bacterial classification AJ07 is mixed according to 1:5 mass ratio with propagating culture medium by the AJ07 culture material activated; Solid fermentation 3 days at 20 DEG C, preferably fermentation 5 days, obtains the fermentation material of bacterial classification AJ07, further, makes the viable count 10 of final fermentation material
10more than cfu/g;
The bacterial classification AJ07 culture material that described activation is good accesses at LB inclined-plane picking strains A J07 to be activated to carry out activating at 25 DEG C in activated liquid LB substratum and obtain; Thus to obtain activating rear cell concentration be 10
10the culture material of cfu/ml; Described activated liquid LB substratum is: peptone 10g/L, NaCl 10g/L, yeast extract paste 5g/L, pH 7.0 ~ 7.5; 121 DEG C, 30min sterilizing;
Described propagating culture medium solid feed and proportioning:
| Humic acid | 55wt％ |
| Groundnut meal | 5wt％ |
| Wheat bran | 3wt％ |
| Wilkinite | 37wt％ |
The humidity of propagating culture medium finished product preferably 55%.
Further, described granulation is dried can conventionally granulation, but the method for preferred following contriver's employing, more excellent effect can be produced:
It is added by compound in stirrer and add binding agent to stir that granulation is dried, and the fully mixing that adds water makes water content lower than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents; Namely obtain seabed sedimentation oil degradation microbial inoculum after cooling, ensure that in seabed sedimentation oil degradation microbial inoculum, viable count reaches 10
9cfu/ml;
Preferably, described following current is dried, and specifically enters in dram by wet granular by the intermediate bin of dryer front portion, meanwhile hot blast enters dram in a co-current manner, under in rotary barrel, flight drives, material is taken up, and forms point material curtain spreading uniform comparatively dense;
Preferably, the raw materials of oil degradation microbe carrier is the fine powder of below 20 orders;
Preferably, the raw materials of propagating culture medium is the fine powder of below 20 orders.
Described dryer can select any one can complete the commercially available dryer of baking step, but the dryer of this model of preferred following manufacturer production: Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
Embodiment 3
Seabed sedimentation oil degradation microbial inoculum is that after being mixed according to the mass ratio of 1:10 with the raw materials of oil degradation microbe carrier by the fermentation material of bacterial classification AJ07, granulation is dried and obtains; Further, final bacteria suspension concentration is made to be 10
10more than cfu/ml is good;
Raw materials and the proportioning thereof of described oil degradation microbe carrier comprise:
| Humic acid | 75wt％ |
| Groundnut meal | 3wt％ |
| Wheat bran | 3wt％ |
| Wilkinite | 18wt％ |
| Na 2HPO 4 | 0.5wt％ |
| KH 2PO 4 | 0.2wt％ |
| NH 4NO 3 | 0.3wt％ |
。
Further, the fermentation material of described bacterial classification AJ07 can adopt any one fermentation process known to ordinary skill in the art to obtain.But the following methods adopting contriver to use produces fermentation material, can obtain better effect:
The fermentation material of bacterial classification AJ07 is mixed according to 1:8 mass ratio with propagating culture medium by the bacterial classification AJ07 culture material activated; At 37 DEG C, solid fermentation 5 days, obtains the fermentation material of bacterial classification AJ07, further, makes the viable count 10 of final fermentation material
10more than cfu/g;
Described bacterial classification AJ07 culture material to access in activated liquid LB substratum 37 DEG C at LB inclined-plane picking strains A J07 to be activated to carry out activating and obtain; Thus to obtain activating rear cell concentration be 10
10the culture material of cfu/ml; Described activated liquid LB substratum is: peptone 10g/L, NaCl 10g/L, yeast extract paste 5g/L, pH 7.0 ~ 7.5; 121 DEG C, 30min sterilizing;
Described propagating culture medium solid feed and proportioning:
| Humic acid | 74wt％ |
| Groundnut meal | 4wt％ |
| Wheat bran | 7wt％ |
| Wilkinite | 15wt％ |
The humidity of propagating culture medium finished product preferably 55%.
Further, described granulation is dried can conventionally granulation, but the method for preferred following contriver's employing, more excellent effect can be produced:
It is added by compound in stirrer and add binding agent to stir that granulation is dried, and the fully mixing that adds water makes water content lower than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents; Namely obtain seabed sedimentation oil degradation microbial inoculum after cooling, ensure that in seabed sedimentation oil degradation microbial inoculum, viable count reaches 10
9cfu/ml;
Preferably, described following current is dried, and specifically enters in dram by wet granular by the intermediate bin of dryer front portion, meanwhile hot blast enters dram in a co-current manner, under in rotary barrel, flight drives, material is taken up, and forms point material curtain spreading uniform comparatively dense;
Preferably, the raw materials of oil degradation microbe carrier is the fine powder of below 20 orders;
Preferably, the raw materials of propagating culture medium is the fine powder of below 20 orders.
Described dryer can select any one can complete the commercially available dryer of baking step, but the dryer of this model of preferred following manufacturer production: Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
Two, strains A J07 oil degradation performance measurement
1) Erlenmeyer flask of 150ml adds ASM substratum 50ml/ bottle 121 DEG C of sterilizing 30min;
ASM culture medium prescription: NaCl:30g, NH
4nO
3: 1g, KH
2pO
4: 0.2245g, Na
2hPO
412H
2o:5g, natural sea-water 1000ml, micro solution: 10ml, pH 7.5.Sterilising temp 121 DEG C, 30min.
Micro solution is filled a prescription:
| CaCl 2 | FeCl.6H 2O | CuSO 4 | MnCl.4H 2O | ZnSO 4·7H 2O | Distilled water |
| 2mg | 50mg | 0.5mg | 0.5mg | 10mg | 1000ml |
2) 250ul crude oil and micro solution 0.5ml is added by the substratum of bacterium of having gone out;
3) cell concentration 10 of bacterial strain is controlled
9cfu/ml, adds 1ml bacteria suspension respectively in Erlenmeyer flask;
4) not add the blank ASM substratum (with the addition of 250ul crude oil) of bacterium for contrast;
5) 15 DEG C of quiescent culture 7d, experiment is in triplicate; Contrast effect as shown in Figure 4;
6) measuring method of petroleum degradation rate:
A: above-mentioned cultured Erlenmeyer flask is taken out add sherwood oil 20ml pour in separating funnel leave standstill 15min.
B: open separating funnel by aqueous phase separation out, leave residual petroleum, after trim, centrifugal 1000r/15min, pours liquid into volumetric flask.
C: by be separated in B the aqueous phase obtained pour into again 15ml sherwood oil carry out 2 times extraction leave standstill 10min repeat B work.
D: get 10ml sherwood oil cleaning separating funnel, and imported volumetric flask.
E: volumetric flask liquid capacity-fixed.To be measured.
The measuring method of petroleum degradation rate: ultraviolet spectrophotometry (wavelength 225nm) measures degradation rate and defines by following formula:
C
0control sample light absorption value, C
1it is working sample light absorption value.Degradation rate is the mean value of three samples.
The petroleum degradation rate of strains A J07
The mensuration of table 1 strains A J07 oil degradation performance
Three, the oil degradation performance measuring and evaluating of strains A J07 microbial inoculum particle
Get the viable count prepared respectively and reach 10
9microbial inoculum particle 0.2g, 0.5g, 1g, 2g, 4g of cfu/ml, be added into respectively in the aseptic triangular flask loading the not petroliferous ASM substratum of 100mL, add the sterilizing oil of 0.5mL, mixing shakes up, and after 15 DEG C of static gas wave refrigerator 9d, measures petroleum degradation rate.With do not add microbial inoculum particle containing oil ASM substratum in contrast.
Table 2 oil degradation bacterial strain AJ07 microbial inoculum particle addition is on the impact (petroleum degradation rate %) of oil degradation performance
Four, microbial inoculum carrier is to the trophism of strains A J07
Test method:
The microbial inoculum particle 1800g that Example 1 method is obtained, be divided into 18 parts, every part of 100g, be placed in withstand voltage Anaerobic culturel bottle, every portion of microbial inoculum adds natural sea-water 200ml, first withstand voltage Anaerobic culturel bottle is made to vacuumize, nitrogen is filled with after exhausted air, pressure is made to reach 1 normal atmosphere, then withstand voltage Anaerobic culturel bottle is placed in 15 DEG C of low temperature incubators and cultivates, within 5 weeks, get weekly 3 bottles respectively at the 1st week-, on LB substratum, carry out viable count counting according to gradient dilution method, calculate weekly the breeding situation of strains A J07.Compare with the viable count in original microbial inoculum.
Experimental result
As shown in Figure 5, along with the prolongation of incubation time, AJ07 viable count is in rising trend gradually, tends towards stability to during 4th week, illustrates that microbial inoculum carrier can provide nutrition needed for growth for strains A J07.
Annex:
Sequence table
SEQ ID No.1 (the 16S rRNA sequence of strains A J07 (Rhodococcus aetherivorans)):
AGGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGGGATTCGCTCCACCTCGCGGTATCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGCGAGTCCCCAACCGAATTGCTGGCAACACAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGAAACCCCATCTCTGGGGCGGTCCGGTGTATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGATCCCGTGGAAGGAAACCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTACCCACGCTTTCGCTCCTCAGCGTCAGTTACTGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTGCAGTACTCAAGTCTGCCCGTATCGCCTGCAAGCCCGCAGTTGAGCTGCGGGTTTTCACAGACGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAGTAATTCCGGACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTGTACCTACCGTCACTCGCGCTTCGTCGGTACTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGGGCCGTTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATCCTGCACCGGAAAACCTTTCCACCCCGGAACATGCATCCCGAGGTCCTATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGGGCAGATCACCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCAGCAAGC
<110> Dalian Nationality College
<120> mono-strain has bacterium AJ07 and its purposes and the seabed sedimentation oil degradation microbial inoculum of oil degradation function
<160> 1
<210> 1
<211> 1344
<212> rDNA
<213> Rhodococcus aetherivorans
<400> 1
AGGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGGGATTCGCTCCACCTCGCGGTATCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGCGAGTCCCCAACCGAATTGCTGGCAACACAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGAAACCCCATCTCTGGGGCGGTCCGGTGTATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGATCCCGTGGAAGGAAACCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTACCCACGCTTTCGCTCCTCAGCGTCAGTTACTGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTGCAGTACTCAAGTCTGCCCGTATCGCCTGCAAGCCCGCAGTTGAGCTGCGGGTTTTCACAGACGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAGTAATTCCGGACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTGTACCTACCGTCACTCGCGCTTCGTCGGTACTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGGGCCGTTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATCCTGCACCGGAAAACCTTTCCACCCCGGAACATGCATCCCGAGGTCCTATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGGGCAGATCACCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCAGCAAGC
Claims (9)
1. a strain has the strains A J07 of oil degradation function, it is characterized in that, its deposit number is CGMCC No.10350, and its 16SrRNA sequence is as shown in SEQ ID No.1.
2. a strain as claimed in claim 1 has the strains A J07 of oil degradation function, Morphology and physiology biochemical character:
1) colony morphology characteristic
LB substratum was cultivated after 7 days, and in orange, rat is opaque, slightly gloss, smooth, and colony edge is neatly smooth;
2) strain morphology and physio-biochemical characteristics thereof
Gram positive aerobic bacterium, catalase and nitrate reductase enzyme positive, edwardsiella hoshinae, not hydrolyzed starch, thalline rod-short, 1.1-1.6*0.5-0.7 μm, all can grow within the scope of NaCl concentration 0-20%, the suitableeest NaCl concentration 3%, the temperature range 10-50 DEG C of growth, optimum temperature range 28-37 DEG C, pH scope 5-10.5, optimal pH 7.5-8.5, can not utilize sucrose, fructose and lactose, can utilize glucose and N.F,USP MANNITOL.
3. a strain as claimed in claim 1 strains A J07 with oil degradation function is carrying out the purposes of improvement of petroleum pollution in the mode of oil degradation.
4. a seabed sedimentation oil degradation microbial inoculum, be a strain according to claim 1 is had the fermentation material of the strains A J07 of oil degradation function and the raw materials of the oil degradation microbe carrier mass ratio according to 1:5-15, after the mass ratio mixing of preferred 1:10, granulation is dried and obtains;
Raw materials and the proportioning thereof of described oil degradation microbe carrier comprise:
。
5. seabed sedimentation oil degradation microbial inoculum as claimed in claim 4, is characterized in that: the fermentation material of strains A J07 is that the mass ratio of preferred 1:10 mixes by the strains A J07 culture material that activate and propagating culture medium according to 1:5-10 mass ratio; Solid fermentation 3 ~ 7 days at 20 ~ 50 DEG C, preferably 37 DEG C, preferably fermentation 5 days and the fermentation material of bacterial classification AJ07;
Described activation good bacterial classification AJ07 culture material be in the LB inclined-plane picking bacterial strain access activated liquid LB substratum being suitable for oil degradation to be activated at 25-50 DEG C, preferably 37 DEG C carry out activating and obtain; Thus to obtain activating rear cell concentration be 10
10cfu/ml culture material; Described activated liquid LB substratum is: peptone 10g/L, NaCl 10g/L, yeast extract paste 5g/L, pH 7.0 ~ 7.5; 121 DEG C, 30min sterilizing;
Described propagating culture medium solid feed and proportioning:
The humidity of propagating culture medium finished product preferably 55%.
6. seabed sedimentation oil degradation microbial inoculum as claimed in claim 4, it is characterized in that: it is added by compound in stirrer and add binding agent to stir that described granulation is dried, the fully mixing that adds water makes water content lower than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents; Namely obtain microbial inoculum after cooling, ensure that in microbial inoculum, viable count reaches 10
9cfu/ml.
7. seabed sedimentation oil degradation microbial inoculum as claimed in claim 6, it is characterized in that: it is enter in dram by wet granular by the intermediate bin of dryer front portion that described following current is dried, meanwhile hot blast enters dram in a co-current manner, under in rotary barrel, flight drives, material is taken up, and forms point material curtain spreading uniform comparatively dense.
8. seabed sedimentation oil degradation microbial inoculum as claimed in claim 4, is characterized in that: the raw materials of described oil degradation microbe carrier is the fine powder of below 20 orders.
9. seabed sedimentation oil degradation microbial inoculum as claimed in claim 4, is characterized in that: the raw materials of described propagating culture medium is the fine powder of below 20 orders.
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| WO2011060048A2 (en) * | 2009-11-11 | 2011-05-19 | Massachusetts Institute Of Technology | Methods for producing polyhydroxyalkanoate copolymer with high medium chain length monomer content |
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