CN105695360B - A kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 and its application - Google Patents
A kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 and its application Download PDFInfo
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Abstract
The invention discloses a kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 and its applications.Isolated one plant of the present invention using phenanthrene as the degradation bacteria strains of carbon source, according to strain morphology, physiological characteristic, Gram-reaction, the analysis of 16S rDNA gene sequencing, DNA-DNA hybrid experiment and Phylogenetic Analysis, identifies the different strains that the bacterial strain is Acinetobacter tandoii kind and be named as Acinetobacter tandoii LJ-5.The bacterium can degrade luxuriant and rich with fragrance using phenanthrene as sole carbon source and soon: be 100-200mgL in luxuriant and rich with fragrance initial concentration‑1After cultivating 3 days under environment, degradation rate is up to 80% or more；It is 400-1000mgL in luxuriant and rich with fragrance initial concentration‑1After cultivating 7 days under environment, degradation rate can reach 60% or more；The bacterial strain has preferable application potential at biological prosthetic aspect.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5
And its application.
Background technique
Polycyclic aromatic hydrocarbon (PAHs) is a kind of environmental pollutants received significant attention, generally existing and continuous in the environment
Accumulation.Luxuriant and rich with fragrance (phenanthrene) is a kind of thrcylic aromatic hydrocarbon, the carcinogenicity of it and PAHs have very close relationship, relies on it
Unique chemical structure, phenanthrene become PAHs research mode compound (Li et al., 2010；Liu Shuan etc., 2013).Due to phenanthrene
With characteristics such as persistence, carcinogenic, teratogenesis, mutagenicity and bioaccumulations, ecological environment and human health can be constituted
Significant damage (Yang Xuan etc., 2012).
The Natural Attenuation of poisonous and harmful organic pollutant relies primarily on related microorganisms metabolism in environment, biological prosthetic
Technology has many advantages, such as that at low cost, effect is good, without secondary pollution, therefore this method is that current luxuriant and rich with fragrance pollution amelioration is most potential repairs
Multiple means (Samanta et al., 1999；Yang Xiuhong etc., 2012).Currently, reported phenanthrene degradation bacteria strain includes
Agmenellum sp.、Aeromonas sp.、Alcaligenes sp.、Acinetobacter sp.、Bacillus sp.、
Berjerinckia sp.、Burkholderia sp.、Corynebacterium sp.、Cyclotrophicus sp.、
Flavobacterium sp.、Micrococcus sp.、Moraxella sp.、Mycobacterium sp.、
Nocardioides sp.、Pseudomonas sp.、Lutibacterium sp.、Rhodococcus sp.、
Streptomyces sp.、Sphingomonas sp.、Stenotrophomonas sp.、Vibrio sp.、
Paenibacillus sp. etc. (Zhao et al., 2008).Recent domestic researcher divides from natural environment
From obtaining some phenanthrene degradation bacteria, and Primary Study (Hou Ning etc., 2013) is carried out to its degradation property.Ni Xue etc. is dirty from polycyclic aromatic hydrocarbon
Contaminate in area's plant Stenotrophomonas category (Stenotrophomonas sp.) that isolated two plants have a luxuriant and rich with fragrance ability of degrading and
Pseudomonas (Pseudomonas sp.), the phenanthrene of two plants of bacterium in 7d in degradable 90% minimal medium
(100mg·L-1) (Ni Xue etc., 2013).Deng Jun etc. is from for a long time by 1 plant of phenanthrene degradation bacteria oxygen isolated in PAHs contaminated soil
Change arthrobacterium (Arthrobacter oxydans), is 50mgL in luxuriant and rich with fragrance initial mass concentration-1Inorganic salts culture solution in cultivate
After 5d, luxuriant and rich with fragrance degradation rate is 60% or so (Deng Jun etc., 2010).The oil-polluted soils acquisition soil near Shengli Oil Field such as Tian Zhigang
Sample, screening can be 200mgL in luxuriant and rich with fragrance initial concentration using phenanthrene as the bacillus of sole carbon source-1When, the phenanthrene after cultivating 96h
Degradation rate is 98.36% (Tian Zhigang etc., 2011).Currently, due to the human activities such as industry and mining, agricultural and soil environment background
It is worth serious pollution and severe overweight that high factor has had resulted in Polycyclic Aromatic Hydrocarbonat Existing in Environment, and chemical garden and periphery soil
Earth, oil-producing region, minery, sewage irrigation area major pollutants be all polycyclic aromatic hydrocarbon (Liu Fang etc., 2011).Therefore, it filters out
The bacterial strain of the high concentration that can effectively degrade phenanthrene has more application value.
Summary of the invention
The first purpose of the invention is to provide a kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5, which is
Activated sludge is acquired from Northern Shandong petroleum waste water treatment plant, is tamed for a long time using high concentration phenanthrene as carbon source, by more
It is secondary to screen and isolate and purify to obtain efficient, the high concentration that degrade phenanthrene phenanthrene degradation bacteria；Acinetobacter tandoii LJ-
5 were preserved in Guangdong Province's Culture Collection (GDMCC) on March 1st, 2016, address: Xianlie Middle Road, Guangzhou City 100
5 building, the building of compound the 59th, Guangdong Microbes Inst, deposit number are as follows: GDMCC No:60010.
The bacteria characteristic of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 of the invention is described as follows:
After the bacterium is activated, grown for 24 hours on the plate made of solid nutrient medium under conditions of 30 DEG C aerobic
Afterwards, can be formed diameter be smooth 0.5-2.0mm canescence, circle, surface, raising upward slightly, opaque, neat in edge,
No gemma, atrichia, Gram's staining are negative, bacterial strain individual is rod-shaped bacterium colony.The bacterium is obligate aerobic bacteria, cell ruler
Very little about 0.7-1.0 × 1.0-1.5 μm.The transmission electron microscope figure of its cell is shown in Fig. 1.
Molecular biological characteristic: the DNA G+C content of the bacterium be 41.0%, this content just fall in it has been reported that cross
Within the scope of where the DNA of bacteria G+C content of acinetobacter；Expand the 16s rRNA gene of the bacterium and be sequenced, obtain as
DNA sequence dna shown in SEQ ID NO.1, using the sequence using ortho position phase connection and minimum Evolve-ment law phylogenetic tree construction
The result shows that the bacterium and Acinetobacter tandoii DSM 14970THomology highest, up to 97.7%；DNA-DNA is miscellaneous
Experiment is handed over to show the bacterium and Acinetobacter tandoii DSM 14970TResults of hybridization is 90.11 ± 0.8%, is higher than miscellaneous
Threshold values 70% is handed over, illustrates that the bacterium is to belong to same kind of Acinetobacter tandoii not in molecular biology level
Same strain, therefore phenanthrene degradation bacteria LJ-5 of the invention is named as Acinetobacter tandoii LJ-5.
The experiment that Acinetobacter tandoii LJ-5 is applied to luxuriant and rich with fragrance degradation shows: at 30 DEG C, pH 7.0 is not added
Under the condition of culture of the liquid nutrient media of NaCl, Acinetobacter tandoii LJ-5 can soon degrade phenanthrene,
It is 100-200mgL in luxuriant and rich with fragrance initial concentration-1Minimal medium culture 3 days after, degradation rate is up to 80% or more；In phenanthrene
Initial concentration in 400-1000mgL-1Minimal medium culture 7 days after, degradation rate can reach 60% or more；Explanation
Acinetobacter tandoii LJ-5 is that a kind of degradation is luxuriant and rich with fragrance and be resistant to the very strong bacterial strain of luxuriant and rich with fragrance ability.Phenanthrene is often as polycyclic
The mode compound of aromatic hydrocarbons, it can thus be anticipated that the bacterium has preferable tolerance and degradability to polycyclic aromatic hydrocarbon.Therefore, originally
Second purpose of invention is to provide phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 answering in degrading polycyclic aromatic hydrocarbons
With, further, application of the phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 in degradation phenanthrene.
Acinetobacter tandoii LJ-5 of the invention be separate and identify in activated sludge it is novel motionless
The bacterial strain of Bacillus has not been reported PAHs Study on degradation about Acinetobacter tandoii both at home and abroad；
Acinetobacter tandoii LJ-5 can effectively be degraded using high concentration phenanthrene as carbon source, which is in luxuriant and rich with fragrance initial concentration
50-200mg·L-1After cultivating 3 days under environment, degradation rate is up to 80% or more；It is 400-1000mg in luxuriant and rich with fragrance initial concentration
L-1After cultivating 7 days under environment, degradation rate can reach 60% or more；The bacterium is that a kind of degradation is luxuriant and rich with fragrance and the luxuriant and rich with fragrance ability of tolerance is very strong
Bacterial strain, it is adaptable to polycyclic aromatic hydrocarbon；Polycyclic aromatic hydrocarbons contaminated environment it is biological prosthetic in have a good application prospect.
Acinetobacter tandoii LJ-5 of the invention is preserved in Guangdong Province's microbial bacteria on March 1st, 2016
Kind collection (GDMCC), address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst are protected
Hiding number are as follows: GDMCC No:60010.
Detailed description of the invention
Fig. 1 is the form of Acinetobacter tandoii LJ-5, wherein (a) is to grow on solid nutrient medium
Acinetobacter tandoii LJ-5 group；It (b) is Acinetobacter tandoii LJ-5 transmission electron microscope
Figure: atrichia, scale bar are 0.5 μm；It (c) is Acinetobacter tandoii LJ-5 transmission electron microscope figure, ratio
Ruler is 1 μm.
Fig. 2 is the Acinetobacter tandoii LJ-5 bacterium related to its constructed using adjacent method based on 16s
The phylogenetic relationship of rRNA gene order, value of bootstrapping setting repeat 1000 times, and value of bootstrapping is shown only in figure and is greater than 50%
As a result, scale bar 0.01 represents the replacement rate of each nucleotide, LJ-5TRepresent Acinetobacter tandoii LJ-5.
Fig. 3 be using minimum Evolve-ment law building Acinetobacter tandoii LJ-5 bacterium related to its based on
The phylogenetic relationship of 16s rRNA gene order, value of bootstrapping setting repeat 1000 times, value of bootstrapping are shown only in figure and is greater than
50% as a result, scale bar 0.005 represents the replacement rate of each nucleotide, LJ-5TRepresent Acinetobacter tandoii
LJ-5。
Fig. 4 is influence of the different growth conditions to Acinetobacter tandoii LJ-5, wherein (a) pH, (b) warm
Degree, (c) tolerance of salinity.
Fig. 5 is that Acinetobacter tandoii LJ-5 grows song in the minimal medium of the phenanthrene containing various concentration
Line, luxuriant and rich with fragrance initial concentration are 100-1000mgL-1。
Fig. 6 is minimal medium China and Philippines degradation feelings of the Acinetobacter tandoii LJ-5 in the phenanthrene containing various concentration
Condition, luxuriant and rich with fragrance initial concentration are 100-1000mgL-1。
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1
1 material
1.1 sample source
Activated sludge is acquired from Northern Shandong petroleum waste water treatment plant, is tamed and dociled for a long time using high concentration phenanthrene as carbon source
Change, obtains efficient phenanthrene degradation bacteria LJ-5 by repeatedly screening and isolating and purifying.
1.2 culture medium
1.2.1 minimal medium
Minimal medium is for degradation experiment luxuriant and rich with fragrance under the conditions of the enrichment culture of microorganism in sample and pure bacterium.The culture medium
Formula is shown in Table 1 (containing luxuriant and rich with fragrance solution).
1 minimal medium formula of table
1.2.2 nutrient medium
The culture of the conventional microbiologicals such as separation, purifying, preservation, activation of the nutrient medium (LB culture medium) for bacterium.
This experiment liquid nutrient media type used and ingredient are shown in Table 2.If experiment needs to prepare solid nutrient medium, it is only necessary to
Increase the agar powder of 1.5-2% on the basis of original culture medium prescription.If the condition of culture of bacterial strain is without special instruction, medium pH
It is adjusted to 7.2.
2 nutrient medium ingredients of table
2 methods
Domestication, screening and the separation of 2.1 bacterial strains
The activated sludge of acquisition is added in minimal medium, is 1000mgL with concentration-1It is luxuriant and rich with fragrance as degrading
Substrate (contains 1000mgL in minimal medium-1Phenanthrene), be placed in 30 DEG C of incubators and be protected from light shake culture.It utilizes
Bacterial strain domestication is carried out by the minimal medium of carbon source of phenanthrene, 7d is an acclimation period；10% inoculum concentration is taken to be transferred to tool
There is the fresh using phenanthrene in the minimal medium of carbon source and to repeat above-mentioned enrichment process of identical cultivating system, so repeatedly three
It is secondary.
The forth generation enrichment culture sample of above-mentioned acquisition is coated separation, sample nutrition culture with dilution-plate method
Base separation.Coated sample is placed under the conditions of former cultivation temperature and is cultivated, by 48 hours or so, media surface formed bright
Aobvious single colonie according to the different several single colonies of the features picking such as form size, color, transparency of bacterium colony, and is being sought
It supports and carries out scribing line purifying on culture medium flat plate, cultivate.If still it is observed that the list of different characteristic on the plate by scribing line purifying
It is carried out scribing line separation, until it can only observe the single colonie of same characteristic features on same plate by bacterium colony again.Experiment
Middle screening obtains 1 plant of bacterial strain LJ-5 for having efficient degradation performance to phenanthrene.The LJ-5 single bacterium of picking after purification falls on respective liquid battalion
It supports culture in culture medium and bacterium solution and sterile glycerol is mixed into packing (glycerol concentration into sterile 2ml cryopreservation tube to logarithmic phase
15%), to be placed in -80 DEG C of long-term preservations.
The morphological feature of 2.2 bacterial strain LJ-5
LJ-5 is one plant of bacterium isolated from activated sludge, after activated, solid under conditions of 30 DEG C aerobic
After being grown for 24 hours on plate made of body nutrient medium, can be formed diameter be 0.5-2.0mm canescence, circle, surface it is smooth,
Raising upward slightly, opaque, neat in edge, be negative without gemma, atrichia, Gram's staining, bacterial strain individual be it is rod-shaped
Bacterium colony.The bacterium is obligate aerobic bacteria, and cell size is about 0.7-1.0 × 1.0-1.5 μm.The transmitted electron of its cell is aobvious
Micro mirror figure is shown in Fig. 1.
The physiological characteristic of 2.3 bacterial strain LJ-5
This experiment tests many LJ-5 physiological properties, these physical signs are mainly the utilization of carbon source for measuring bacterial strain LJ-5
And produce acid etc..Physiological property is listed in Table 3.Utilization of carbon source, production acid and other tests use API ID 32GN and API
20NE microbial identification kit tests (Biomerieux SA).
The physiological property of 3 bacterial strain LJ-5 of table
Note :+, it is positive；W, weakly positive；, negative.
The molecular biological characteristic of 2.4 bacterial strain LJ-5 is identified
Molecular biological characteristic identification mainly includes the research of three aspects, DNAG+C assay, separated bacterial strain LJ-
DNA-DNA hybrid experiment, sequencing and the building of phylogenetic tree between 5 and mode bacterium.These experiments can classify for bacterium
Positioning on provides scientific evidence.
2.4.1DNA G+C assay
The measurement of DNA G+C content mainly applies to high performance liquid chromatography, and specific steps refer to Mesbah et al.
(1989) report.The DNA G+C content that measurement obtains LJ-5 is 41.0%, this content is just fallen in it has been reported that crossing not
(38.1-54.7% within the scope of where the DNA of bacteria G+C content of lever Pseudomonas；https:// olive.broadinstitute.org/)。
2.4.2 sequencing and the building of phylogenetic tree
Before carrying out sequencing and phylogenetic tree construction, it is necessary first to extract DNA (the bacterium base that experiment uses of bacterium
Because group DNA rapidly extracting kit comes from Beijing Ai Delai Biotechnology Co., Ltd).In order to which the taxology to bacterium is ground
Study carefully, it usually needs amplification 16s rRNA gene and phylogenetic tree construction, the gene of amplification are to encode rRNA in prokaryotes
Section of DNA in institute's component part, because of its conservative with height, specificity and appropriate sequence length, usually by with
In detection and identification bacterium.
Polymerase chain reaction (PCR) is primarily used to expand different genetic fragments, and PCR is needing different primers (just
To being respectively 27F and 1492R with reverse primer；Baker et al., 2003), the system of pcr amplification reaction: 10 × buffer
2.5 μ l, Mg2+(25mmol/l) 1.5 μ l, dNTP (25mmol/l) 0.3 μ l, 0.5 μ l of forward primer (10mmol/l), reverse primer
(10mmol/l) 0.5 μ l, 0.25 μ l, DNA group template of Taq enzyme 0.1 μ l, 19.35 μ l of deionized water.Pcr amplification reaction condition: 95
It is denaturalized under the conditions of DEG C, anneals under conditions of 55 DEG C, extend under conditions of 72 DEG C, under conditions of this process recycles 30 times, 72 DEG C
Extend 10min, PCR is saved under the conditions of 4 DEG C after reaction.With the agarose of 0.75-1% and addition after gene needed for expanding
Nucleic acid staining agent GelRed is configured to gel piece, and PCR product and the DNA marker comprising various long fragments are added in gel piece
Object (maker) is simultaneously placed in electrophoresis apparatus, and TBE (Tris boric acid) buffer is packed into electrophoresis apparatus, makes electrophoresis apparatus in certain electricity
It is taken out after pressure work 20min, is placed under the ultraviolet lamp of 300nm and is observed to determine the success of PCR product amplified reaction.So
Successful PCR product will be expanded afterwards and is sent to the sequencing of Hua Da Gene Tech. Company Limited, and sequencing primer is identical as amplimer.Pass through
The LJ-5 16s rRNA gene order for a length of 1437bp that PCR product progress gene sequencing is obtained, sequence such as SEQ
Shown in ID NO.1.
By the 16s rRNA gene order that resulting bacterium LJ-5 is sequenced upload to EzTaxon-e (http://eztaxon- e.ezbiocloud.net/；Kim et al., 2012) on, this website can be by the sequence of submission and the typical bacterium being recognized kind
The 16s rRNA gene order of strain is compared, and obtains the similarity information between sequence.It is according to the interpretation of result of sequence alignment
Mode bacterium of the corresponding typical strain as this experiment isolated strains can be chosen, while can be with the 16s rRNA of acquisition model bacterium
Gene order constructs Phylogenetic Analysis to prove that mode bacterium and experiment isolated strains LJ-5 have difference, thus to identify point
From bacterial strain LJ-5.Phylogenetic tree construction generallys use adjoining using 5.05 program of MEGA (Tamura et al., 2011)
Method, minimum Evolve-ment law and maximum parsimony method construct chadogram, and most common of them is adjacent method, and value of bootstrapping usually is set as repeating
Calculate (Felsenstein et al., 1985) 1000 times.Using LJ-5 16s rRNA gene order and with its similarity compared with
High 16s rRNA gene order manufacturing system develops tree, thus obtain LJ-5 16s rRNA gene and its similitude it is higher
Homology result between 16s rRNA gene.Phylogenetic tree using ortho position phase connection and minimum Evolve-ment law building is shown in Fig. 2
With 3, as a result all show LJ-5 and Acinetobacter tandoii DSM 14970THomology highest, up to 97.7%.
2.4.3DNA-DNA hybrid experiment
DNA-DNA hybridization is to be higher than 97% just in the 16s rRNA alignment similarity of mode bacterium and isolated bacterial strain LJ-5
It carries out, it is to prove that the two belongs to two not of the same race or same kind of different variations in molecular biology level
Body.The measurement of DNA-DNA hybrid experiment has used bright biotinylated probe method, and every group of DNA-DNA hybrid experiment needs to carry out
To guarantee the accuracy of experimental data, each pair of bacterium of phase mutual cross need to carry out orthogonal and two groups of experiments of reciprocal cross, tool for three repetitions
Body step is carried out referring to the description of Ezaki et al. (1989).DNA-DNA hybrid experiment refers in molecular biology level,
The method for being often used for distinguishing the higher bacterium of 16s rRNA gene similitude, reliability is also higher than traditional method, LJ-5
Cause and the 16s rRNA gene similitude of its one plant of mode bacterium have been above 97%, therefore it is needed and Acinetobacter
tandoii DSM14970T(97.7%) DNA-DNA hybrid experiment is carried out, experimental result is as follows: LJ-5 and Acinetobacter
tandoii DSM14970TResults of hybridization is 90.11 ± 0.8%, these results are higher than hybridization threshold values 70%, when cross experiment knot
Fruit can then illustrate that two kinds of bacteriums of phase mutual cross are to belong to same kind in molecular biology level when being higher than this threshold values
Different variants.It can show that this tests separated bacterium LJ-5 as Acinetobacter tandoii from the above results
The different strains of kind.Thus the bacterial strain LJ-5 of efficient degradation performance is named as Acinetobacter phenanthrene for what is be separated to
tandoii LJ-5。
The growth conditions of 2.5 bacterial strain LJ-5
The measurement of growth temperature: liquid nutrient media needed for configuration bacterial strain LJ-5 growth takes autoclave after preparing
It sterilizes.Activated bacterial strain LJ-5 is linked into culture medium (experimental group), with the culture medium of not inoculated bacteria as right
According to (control group), culture medium is put under different temperatures and cultivates 7d, control group and the corresponding experimental group of each temperature it is equal there are three
It repeats, all needs the growing state of observation bacterium daily, when encountering the result that naked eyes are difficult to differentiate between, with visible-ultraviolet spectrometry light
Light absorption value of the degree meter measurement culture medium at wavelength X=600nm, finally show that LJ-5 can growth temperature and optimum growth temperature model
It encloses.Test temperature is as follows: 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 42 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and
60℃.As a result as shown in Figure 4 b, in liquid nutrient media, bacterial strain LJ-5 can be grown under the conditions of 25-40 DEG C of temperature,
Optimum growth temperature is 30 DEG C of enrichment temperature of the bacterium.
Grow the measurement of pH: liquid nutrient media needed for configuration bacterial strain LJ-5 growth is adjusted with following buffer system
The pH of culture solution, pH 4.0-5.0,0.1mol/l sodium citrate and 0.1mol/l citric acid；PH 6.0-8.0,0.1mol/l
NaOH and 0.1mol/l KH2PO4；PH 9.0-10.0,0.1mol/l NaHCO3With 0.1mol/l Na2CO3；PH 11.0,
0.1mol/l NaOH and 0.05mol/l Na2HPO4(Zhang et al.,2009).Bacterial strain LJ-5 is linked into liquid nutritional training
It supports in base, each pH does three repetitions, uses the culture medium of not inoculated bacteria as control, liquid nutrient media is put into new bacterium
7d is cultivated at 30 DEG C of optimum temperature of growth, all needs the growing state of observation bacterium daily, when encountering the result that is difficult to differentiate between of naked eyes
When, with visible-light absorption value of the ultraviolet specrophotometer measurement culture medium at wavelength X=600nm, finally show that bacterial strain LJ-5 can
Grow pH and the most suitable growth pH range.The pH of test is as follows: 4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.2,7.5,8.0,
8.5,9.0,9.5,10.0,10.5,11.0.As a result as shown in fig. 4 a, bacterial strain LJ-5 can give birth under the conditions of the pH of 5.0-9.0
Long, the most suitable growth pH is 7.0.
Salinity tolerance: liquid nutrient media needed for configuration bacterial strain LJ-5 growth adjusts the salinity of culture medium.It will
Activated bacterial strain LJ-5 is linked into sterilized liquid nutrient media, and each salinity does three repetitions, with not connecing bacterium
Culture medium as control, by liquid nutrient media be placed on LJ-5 growth optimum condition (30 DEG C, pH7.0) under cultivate 7d,
The growing state for all needing observation bacterium daily is surveyed when encountering the case where naked eyes are difficult to differentiate between with visible-ultraviolet specrophotometer
Determine light absorption value of the culture medium at wavelength X=600nm, preferably obtains the salt concentration range that new bacterium is resistant to.Test salinity
(mass fraction) is as follows: 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.As a result as illustrated in fig. 4 c, bacterium
The salt tolerance of strain LJ-5 is weaker, can grow under conditions of salinity is 0% to 3%, and the growing way under 0% salinity
It is best.
The growth and degradation of bacterial strain LJ-5 under 2.6 different luxuriant and rich with fragrance concentration conditions
According to the above experimental result, determine that the best growing condition of bacterial strain is 30 DEG C of temperature, pH 7.0 does not add NaCl.
Growth and degradation experiment of the bacterial strain LJ-5 in different luxuriant and rich with fragrance concentration carry out under this condition.The bacterium of logarithmic growth phase will be in
Strain LJ-5 by 10% inoculum concentration (the absorbance OD of original bacteria liquid be 0.20, original bacteria liquid is obtained according to classic flat-plate counting method and is contained
Cell number be 1.1*107CFU·mL-1) to be inoculated in respectively containing initial luxuriant and rich with fragrance concentration be 100,200,400,600,800,
1000mg·L-1Minimal medium in, shaken cultivation, and doing parallel laboratory test 3 times.
The measurement of luxuriant and rich with fragrance concentration uses ultraviolet spectrophotometry, i.e., hexamethylene extraction is added in pretreated luxuriant and rich with fragrance solution, filled
Divide after shaking to be transferred in separatory funnel and stand.Supernatant is taken after layering, and lower liquid is put back to shaking flask with isometric hexamethylene weight
Multiple extraction, combining extraction liquid are filtered with anhydrous sodium sulfate, and then revolving, nitrogen are put into 10mL volumetric flask after blowing, fixed with hexamethylene
Hold.This experiment measures absorbance using hexamethylene as blank control at 251nm wavelength.
The measurement of microbial cell concentration uses photoelectric turbidimetry, is indicated with OD, i.e., ultraviolet light is saturating when wavelength is 600nm
Cross the OD value of measured bacteria liquid sample.
The growth curve of bacterial strain LJ-5 is as shown in Figure 5.The results show that LJ-5 can be grown under the conditions of high concentration phenanthrene.Root
Show that bacterial strain LJ-5 can soon degrade luxuriant and rich with fragrance (Fig. 6) according to determined by ultraviolet spectrophotometry and analysis, in luxuriant and rich with fragrance initial concentration 100
To 200mgL-1Inorganic salts culture solution in cultivate 3 days after, degradation rate can reach 80% or more；In luxuriant and rich with fragrance initial concentration 400
To 1000mgL-1Inorganic salts culture solution in cultivate 7 days after, degradation rate can reach 60% or more；Illustrate that bacterial strain LJ-5 is one
Kind can degrade luxuriant and rich with fragrance and be resistant to luxuriant and rich with fragrance very capable bacterial strain, adaptable to polycyclic aromatic hydrocarbon.
Claims (2)
1. a kind of phenanthrene degradation bacteria Acinetobacter tandoii LJ-5, deposit number are as follows: GDMCC No:60010.
2. application of the phenanthrene degradation bacteria Acinetobacter tandoii LJ-5 described in claim 1 in degradation phenanthrene.
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