CN104498411A - Caulobacter T5M6 with selenium removal capability and applications - Google Patents
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Abstract
The invention belongs to the technical field of environment microorganisms, and particularly relates to a caulobacter T5M6 with selenium removal capability and applications thereof in plant selenium-enriching aspect. The caulobacter T5M6 with oxidization action to chemical elemental selenium is characterized by being separated from selenium-polluted soil. Through the strain, insoluble low-valent selenium in chemical elemental selenium and selenium mineral powder can be oxidized into soluble high-valent selenium in bacteria culture medium and soil, and the soluble high-valent selenium can be utilized and enriched by plants, so that the purpose that the microorganisms promote the selenium enrichment can be achieved. The strain is named as Caulobacter sp.T5M6, belongs to a selenium oxidization bacteria and has the preservation number of CCTCC NO:M2014647. Primary study of potted experiments shows that the strain has favorable application prospects in the aspects of microorganisms in promoting the plant selenium enrichment.
Description
Technical field
The invention belongs to technical field of environmental microorganism, be specifically related to a strain has oxygenizement handle bacillus T5M6 and purposes to elemental selenium, described handle bacillus is categorized as the ability that insoluble elemental selenium and selenium mineral powder can be changed into solvable high price selenium by this bacterium of Caulobacter sp.T5M6 in pure culture and soil, meanwhile, this bacterium can also promote that tobacco and Plantula Brassicae chinensis are to the enrichment of selenium.
Background technology
Selenium is a kind of teleorganic trace element, is the necessary component of selenocystein and selenoenzyme, plays a significant role in anticancer, anti-oxidant etc., and appropriate benefit selenium contributes to improving body immunity, and lacks selenium and can cause numerous disease, as Keshan disease etc.Occurring in nature, selenium mainly exists with 4 kinds of valence states: Se (-2), Se (0), Se (+2) and Se (+4), wherein selenite (+4) and selenate (+6) are the most easily absorbed by plants, and the selenium in plant effectively can be absorbed by human body.The soil of China 72% lacks selenium (≤0.6mg/kg), belongs to and lacks the serious country of selenium.WHO and FAO advises human body selenium intake μ g every day 30 ~ 55.From global range, lack selenium crowd (daily intaking amount 7 ~ 11 μ g) and reach 5-10 more than hundred million, lack selenium crowd far away more than the crowd that selenium is excessive.Therefore, selenium is mended by food significant.
The conversion that Sarathchandra etc. (1981) and Dowdle etc. (1998) illustrate selenium the earliest respectively on Science and Environ.Sci.Technol be unable to do without the effect of microorganism.Report the article of a section up to a hundred in micro-reduction selenate and selenite at present, selenium reduction mechanism obtains partial interpretation, and the article reported in low price selenolite rarely has report, the research of selenium oxidation bacteria and application aspect also has no relevant patent application.
The mode of the rich selenium of plant is made to be chemical mode by adding selenite or foliage spray selenite in soil at present, but these mode costs are high, the rich selenium efficiency of plant is general, unsustainable, and can cause poisoning to operator and cause the water pollutions such as river.And the research of the mode of the rich selenium of plant yet there are no report to utilize microorganism can not be utilized by plant the conversion of form selenium that form selenium can be utilized to promote, which cost is low, easy to operate, and poisoning and river will be caused to pollute to operator, rich selenium efficiency comparison is high and stable, and has certain sustainability.
Summary of the invention
The object of the invention is overcome the defect of prior art and fill up the blank of the geographical chemical cycle selenolite aspect of selenium, is separated the handle bacillus T5M6 obtaining a strain and have elemental selenium oxidation capacity and can promote the rich selenium of the plant such as tobacco.Utilize this bacterium can study the mechanism of selenolite and be applied to promotion farm crop Enriching Selenium element in the middle of farm crop, overcome existing plant technique of enriched selenium defect.
This invention finds a strain bacterium, has the ability insoluble zeroth order elemental selenium being converted into high price soluble state selenium.Chemical elemental selenium and selenium mineral powder insoluble low price selenolite can be changed into solvable high price selenium by this bacterial strain in bacteria culture medium and soil, and solvable high price selenium by plant utilization and enrichment, thus can reach the object that microorganism promotes the rich selenium of plant.Identify that it is handle bacillus T5M6 (Caulobacter sp.T5M6), this belongs to is a genus of chemoorganotrophy type, is strict aerobic bacteria, is grown in the water of dystrophy and the poor soil of organic matter.This selenolite bacterial strain T5M6 is separated from enshi high-Se soil.
The present invention is achieved through the following technical solutions:
The present inventor is separated from Chinese Enshi State of Hubei Province high-Se soil, screen the handle bacillus T5M6 that a strain has elemental selenium oxidation capacity and can promote the rich selenium of the plant such as tobacco in soil, applicant is by this bacterium called after handle bacillus T5M6, Caulobactersp.T5M6, China is delivered on December 16th, 2014. Wuhan. Wuhan University's China typical culture collection center (CCTCC) preservation, its preserving number is CCTCC NO:M2014647.
Chemical elemental selenium and selenium mineral powder insoluble low price selenolite can be changed into solvable high price selenium by this bacterial strain in bacteria culture medium and soil, and solvable high price selenium by plant utilization and enrichment, thus can reach the object that microorganism promotes the rich selenium of plant.Shown by potted plant experiment preliminary study, bacterial strain of the present invention promotes to have a good application prospect in the rich selenium of plant in microorganism.
Advantage of the present invention is as follows:
(1) strain that the present invention is separated has the handle bacillus T5M6 that the chemical elemental selenium of zeroth order can be oxidized to high price selenium, fills up the shortage of existing selenium oxidation bacteria.
(2) the insolubility selenium of elemental selenium and selenium mineral powder can be converted into soluble selenium by handle bacillus T5M6 of the present invention in soil, is conducive to plant absorption.
(3) the present invention is that microorganism promotes that the mode of the rich selenium of plant provides a kind of new way of more environmental protection for the rich selenium of plant.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of handle bacillus T5M6 (Caulobacter sp.) the genome 16S rRNA gene that the present invention is separated.
Fig. 1: the handle bacillus T5M6 bacterial strain that the present invention is separated is at the evolutionary degree figure of phylogenetic tree.
Fig. 2: be Technology Roadmap of the present invention.
The result figure of Fig. 3: handle bacillus T5M6 of the present invention oxidizing simple substance selenium in 1/5LB substratum.Description of reference numerals: the T5M6 in Fig. 3 represents the process contrast only adding handle bacillus T5M6 and do not add selenium, CK+Se represents the process contrast only adding selenium and do not add handle bacillus T5M6, and T5M6+Se represents the experiment process not only adding handle bacillus T5M6 but also add selenium;
Fig. 4: the result figure being handle bacillus T5M6 oxidizing simple substance selenium and selenium mineral powder in soil water experiment.Fig. 4 A is the content that in handle bacillus T5M6 soil, oxidation chemistry elemental selenium increases soluble state selenium; Fig. 4 B is the content that in handle bacillus T5M6 soil, selenium oxide breeze increases soluble state selenium.Description of reference numerals: Fig. 4 A is that handle bacillus T5M6 is at the soil water experiment adding chemical elemental selenium process, description of reference numerals: CK result is 0.26 ± 0.02mg/kg (soil), CK+Se result is 2.38 ± 0.56mg/kg (soil), and Se+T5M6 result is 7.61 ± 0.68mg/kg (soil).Fig. 4 B. is that handle bacillus T5M6 is at the soil water experiment adding selenium mineral powder process, CK result is 0.26 ± 0.02mg/kg (soil), CK+Se result is 0.77 ± 0.15mg/kg (soil), and Se+T5M6 result is 3.11 ± 0.65mg/kg (soil).CK represents the blank not adding selenium and do not add handle bacillus T5M6, and CK+Se represents the positive control adding selenium and do not add handle bacillus T5M6, and Se+T5M6 represents the experiment process adding selenium and add handle bacillus T5M6.
Fig. 5: after adding handle bacillus T5M6 process, tobacco plant absorbs the design sketch of selenium.
Fig. 5 A. is Potted tobacco leaf portion selenium content, and Fig. 5 B. is Potted tobacco stem selenium content, and Fig. 5 C. is Potted tobacco root selenium content; Description of reference numerals: CK represents the blank not adding selenium and do not add handle bacillus T5M6, T5M6 represents the control treatment adding handle bacillus T5M6 and do not add selenium, CK+Se represents the positive control adding selenium and do not add handle bacillus T5M6, and Se+T5M6 represents the experiment process adding selenium and add handle bacillus T5M6.
Fig. 6: the selenium content being handle bacillus T5M6 each position after warmhouse booth potted plant experiment promotes the rich selenium of Chinese cabbage plant.Fig. 6 A is potted plant Chinese cabbage over-ground part selenium content; Fig. 6 B is potted plant Plantula Brassicae chinensis underground part selenium content.Description of reference numerals: CK represents the blank not adding selenium and do not add handle bacillus T5M6, and T5M6 represents the control treatment adding handle bacillus T5M6 and do not add selenium, and CK+Se represents the positive control adding selenium and do not add bacterium, and Se+T5M6 represents the experiment process adding selenium and add bacterium.
Embodiment
Embodiment 1: the separation of handle bacillus T5M6 (Caulobacter sp.), qualification
(1) sample is taked: applicant and in July, 2012 collect the present invention's high selenium earth sample used from the topsoil of Hubei Province's Enshitujianationalitymiaonationality Autonomous Prefecture height selenium area.
(2) thalline separation and purification: take soil sample 10g in the triangular flask that 90ml stroke-physiological saline solution is housed, puts in 28 DEG C of shaking tables and vibrates half an hour, then gets 1ml successively and join in 9ml stroke-physiological saline solution and be progressively diluted to 10
-2, 10
-3, 10
-4, 10
-5, get 0.1ml respectively and be coated with in 1/10LB solid medium, each gradient is coated with 3 flat boards, put 28 DEG C of constant incubators to cultivate one week, picking list bacterium colony line purifying, by 50% glycerine after purifying: bacterium liquid (volume ratio 1:1) is stored in EP pipe ,-80 DEG C of preservations.
(3) screening of selenium oxidation bacteria: take 0.1g simple substance selenium powder (CP) in the triangular flask of the 250ml containing 100ml 1/10LB liquid nutrient medium at 115 DEG C of sterilizing 20min, after cooling, access bacterium liquid 150 μ l, 28 DEG C, 8d cultivated by 160rpm shaking table, middle spot sampling (supernatant), by gained supernatant liquor in the centrifugal 10min of 8000rpm, uses atomic fluorescence to carry out detecting selenous content.If tetravalence selenium content obviously increases, illustrate that this bacterium has oxidisability to elemental selenium.
(4) taxonomic identification of selenium oxidation bacteria: use the mono-clonal of each bacterial strain of toothpick picking in 50 μ L aseptic double-distilled waters, mixing.Obtain pcr amplification template with colony polymerase chain reaction (PCR) method, amplification system is 50 μ L systems, universal primer 27F (5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'GGYTACCTTGTTACGACTT3').Amplification condition is: denaturation, 95 DEG C, 5min; Sex change, 94 DEG C, 45sec; Annealing, 49 DEG C, 45sec; Extend, 72 DEG C, 1.5min sex change-extension 35 circulation; Extend: 72 DEG C, 10min.Obtain the nucleotide sequence as shown in sequence table SEQ ID NO:1, sequence length is 1395bp.PCR primer is detected with the agarose gel electrophoresis that concentration is 1%.Deliver to order-checking company (Kechuang true tumor Science and Technology Ltd. is held up in Wuhan) to check order.The 16S rRNA gene order obtained (sequence as shown in SEQ ID NO:1) and existing sequence in GenBank library are compared, the two strain bacterium that the T5M6 bacterial strain similarity that discovery is separated with the present invention is the highest are respectively Caulobacter segnis ATCC 21756 (T) and Caulobacter vibrioides CB51 (T), similarity is respectively 98.24% and 98.10%, and applicant identifies that this bacterium is Caulobacter.The evolutionary degree of this bacterium phylogenetic tree in similar strain by N-J Algorithm Analysis, result as shown in Figure 1.
The mycology feature of handle bacillus (Caulobacter sp.) T5M6:
Bacterium colony is yellow, and neatly smooth, convex surface, opaque, bacterium colony size: less, according to the 16S rRNA of this bacterium, applicant identifies that this bacterial strain is handle bacillus T5M6, and Classification And Nomenclature is Caulobacter sp.
Embodiment 2: the elemental selenium oxidation curve of handle bacillus T5M6
Concrete steps are as described below:
(1) 3 bottles of sterilized 1/10LB liquid nutrient mediums are prepared, every bottle of 100ml.Get three bottles and respectively add the sterilized elemental selenium of 0.1g.Inoculation logarithmic phase (OD600 value about 1.0) T5M6 bacterium liquid, inoculum size 0.25%.Sample 2ml at once after mixing, this sample, as sampling first, is placed in clean centrifuge tube.Each triangular flask is placed in 28 DEG C, cultivates in 160rpm constant-temperature table.Sample time is respectively 0d, 1d, 2d, 4d, 5d, 6d, and this Setup Experiments three repeats.Sampling terminates rear sample and does following process;
(2) the centrifugal 10min of the sample 2ml at every turn got, 12000rpm, retain supernatant liquor, supernatant is in 4 DEG C of Refrigerator stores;
(3) before test sample, get 1ml supernatant in 10ml centrifuge tube, then add 4ml deionized water dilution mixing;
(4) content of Selenium In Some Selenium-rich Biological Samples is detected with Beijing Jitian Instrument Co., Ltd.'s Atomic Fluorescence Spectroscopy (AFS) (HG-AFS), current-carrying is 2.4mol/L HCL (G.R), and reductive agent is 0.5%KOH (A.R)+2%KBH4 (G.R);
(5) to record selenium concentration (μ g/L) for ordinate zou, the time is that X-coordinate builds elemental selenium oxidation graphic representation (as shown in Figure 3).
Embodiment 3: utilize soil water experiment to check handle bacillus T5M6 to the oxidation of elemental selenium and selenium mineral powder
In the present embodiment, the selenium content of selenium mineral powder is 189mg/kg, elemental selenium reagent (chemical pure, 99%);
(1) process in early stage of soil sample: choose prepartion of land, surperficial weeds growing way is even, the native region of unpolluted health, removes top layer weeds, gathers the soil that about 5-10cm is dark, then carries out air-dry, mill, and crosses 40 mesh sieves, collects and continue to employ;
(2) prepare 6 250ml size black cases, each box is weighed into 50g wind desiceted soil, gets three boxes and respectively adds 0.5g selenium mineral powder, gets three boxes and respectively adds 0.1g elemental selenium, mix thoroughly, then in every box, add 10ml deionized water, balance 24h.Test each process and establish three repetitions;
(3) after having balanced, evenly at native top layer flushing liquor, bacterium liquid measure converts according to >1.0 × 108cfu/g (soil), adds the process of handle bacillus T5M6 in the present embodiment, and usage quantity is 1ml handle bacillus T5M6 stoste; The process aliquots of deionized water not adding handle bacillus T5M6 replaces, and after mixing thoroughly, takes weight and record, and be then placed in 28 DEG C of thermostatic chambers and cultivate 1 month, period calculates amount of water according to weighing, and keeps the water ratio that soil is constant;
(4) after having cultivated, by the soil of cultivation 70 DEG C of (temperature can not be too high) oven for drying, mill, cross 100 objects sieves, collect and obtain soil sample to be measured;
(5) " the Selenium in Soil form extracting method " reported according to (2004) such as second lieutenants Wu extracts the content of soluble state selenium in soil sample to be measured and the selenium sample of process extraction continuously;
(6) Beijing Jitian Instrument Co., Ltd.'s Atomic Fluorescence Spectroscopy (AFS) (HG-AFS) detects the content of selenium in soil sample to be measured;
(7) by tested result data SPSS Statistics17.0 statistical software process (http://www.ddooo.com/softdown/53379.htm), with pedological unit weight soluble selenium content (mg/kg
(soil)) be ordinate zou, each is treated to X-coordinate, and Origin 8 software is figure, and result is as shown in Figure 4.
Embodiment 4: handle bacillus T5M6 is promoting the application in tobacco selenium-rich
The selenium content of selenium mineral powder is in the present embodiment 189mg/kg, test plant tobacco (flue-cured tobacco cultivars is No. 1, cloud and mist).
(1) soil pretreatment: 15kg/ basin of being weighed by wind desiceted soil, base manure (composite fertilizer N is in the composite fertilizer then applying selenium mineral powder 396.8g/ basin (5mg/kg (soil)) and 40g/ basin
2o:P
2o
5: K
2o 10:10:20), mix thoroughly, add water by 15% water ratio, balance 3d;
(2) after having balanced, the mother bacterial liquid dilute with water preserved with physiological saline (by bacterium amount >10
7cfu/g
(soil)after adding engagement, native water ratio is 30%), equably bacterium liquid is sprayed at soil surface, then mixes thoroughly, balance 4d (every day sprays a small amount of water, keeps native moistening), each process 10 repetition;
(3), after having balanced, the cigarette seedling (cigarette seedling cultivates time about 30d) cultivated is transplanted;
(4) press tobacco plant growth demand after transplanting and keep sufficient moisture;
(5), after 50d, add bacterium of topdressing and (topdress as the above-mentioned composite fertilizer of 20g/ basin, chase after bacterium amount by >10
6cfu/g
(soil)be appended in the middle of every basin), when sample and carry out first time sampling (comprising plant complete stool sample and pedotheque);
(6), after 90d, tobacco maturation (tobacco gather before) carries out second time sampling (comprising plant complete stool sample and pedotheque);
(7) after plant sample being completed according to conventional tea-manufacturing technology, dry (to water ratio less than 5%), pulverizing, Plant samples is cleared up by the method for measuring (Ministry of Health of the People's Republic of China issues in 2010.03.26, and 2010.06.01 implements) of National Standard of the People's Republic of China GB 500993-2010 selenium in food; After plantation, pedotheque extracts the form selenium of each form selenium and process extraction according to " Selenium in Soil form extracting method " that (2004) such as second lieutenants Wu are reported
(8) selenium in Plant samples and the selenium in soil is measured with Beijing Jitian Instrument Co., Ltd.'s Atomic Fluorescence Spectroscopy (AFS) (HG-AFS);
(9) by tested result data SPSS Statistics17.0 statistical software process (http://www.ddooo.com/softdown/53379.htm), with plant units weight selenium content (mg/kg) for ordinate zou, each is treated to X-coordinate, Origin 8 software is figure, and result as shown in Figure 5.
Embodiment 5: handle bacillus T5M6 is promoting the application in the rich selenium of Chinese cabbage
In the present embodiment, the selenium content of selenium mineral powder is 189mg/kg, and the kind of application is Chinese cabbage
(1) soil pretreatment: 15kg/ basin of being weighed by wind desiceted soil, then applies selenium mineral powder 396.8g/ basin (5mg/kg (soil)), mixes thoroughly, add water by 15% water ratio, balance 3d;
(2) after having balanced, the handle bacillus T5M6 mother liquor dilute with water (proportioning of physiological saline and handle bacillus T5M6 of preserving with physiological saline, or according to the absolute magnitude definition of T 5M6 containing bacterium, by adding bacterium liquid (because bacterial content is unclear in this bacterium liquid, please illustrate with method for normalizing, as cfu/g) often basin soil water ratio is 30% afterwards), equably handle bacillus bacterium liquid is sprayed at soil surface, then mix thoroughly, balance 4d (every day sprays a small amount of water, keeps native moistening).Each process 4 repetition;
(3), after having balanced, Brassica rapa L Plantula Brassicae chinensis seed is sprinkled;
(4) sufficient moisture is kept by Chinese cabbage growth demand after having spread kind;
(5) after 30d, remove unnecessary seedling, retain 3 strains;
(6) after 60d, Chinese cabbage is in growth animated period, and carry out first time sampling (plant complete stool), two strains got by every basin;
(7) after 75d, Chinese cabbage starts cored, gets second time sample (plant complete stool and pedotheque);
(8) sample completes according to after conventional tea-manufacturing technology, oven dry (to water ratio less than 5%), pulverizing, Plant samples is cleared up by National Standard of the People's Republic of China GB 500993-2010 " method for measuring of selenium in food " (Ministry of Health of the People's Republic of China issues in 2010.03.26, and 2010.06.01 implements); After plantation, pedotheque extracts the form selenium of each form selenium and process extraction according to " Selenium in Soil form extracting method " that (2004) such as second lieutenants Wu are reported
(9) selenium in Plant samples and the selenium in soil is measured with the Atomic Fluorescence Spectroscopy (AFS) (model is HG-AFS) that Beijing Jitian Instrument Co., Ltd. produces
(10) by tested result data SPSS Statistics17.0 statistical software process (http://www.ddooo.com/softdown/53379.htm), with plant units weight selenium content (mg/kg) for ordinate zou, each is treated to X-coordinate, Origin 8 software is figure, and result as shown in Figure 6.
Leading reference
1. second lieutenant Wu etc., the research of the form continuous extraction method of Selenium in Soils. soil (Soils), 2004,36 (1): 92 ~ 95
2.Sarathchandra S U,Watkinson J H.Oxidation of elemental selenium to selenite by Bacillusmegaterium[J].Science,1981,211:600-601.
3.Philip R.Dowdle and Ronald S.Oremland.Microbial Oxidation of Elemental Selenium in SoilSlurries and Bacterial Cultures.Environ.Sci.Technol.1998,32,3749-3755
4. National Standard of the People's Republic of China GB 5009.93-2010, the method for measuring of selenium in food, Ministry of Health of the People's Republic of China, issues on 03 26th, 2010, within 06 01st, 2010, implements.
Claims (4)
1. handle bacillus T5M6 (Caulobacter sp.) T5M6 elemental selenium to oxygenizement of a strain separation, be deposited in China typical culture collection center, its preserving number is CCTCC NO:M2014647.
2. handle bacillus T5M6 elemental selenium to oxygenizement of a strain separation as claimed in claim 1, is characterized in that the 16S rRNA gene order of this bacterium is as shown in sequence table SEQ ID NO:1.
3. the application of handle bacillus T5M6 in scarce selenium environment elemental selenium to oxygenizement described in claim 1 or 2.
4. application as claimed in claim 3, is characterized in that, described selenium is zeroth order selenium.
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| WO2020173941A1 (en) | 2019-02-27 | 2020-09-03 | Vib Vzw | Novel plant growth promoting microbial compositions |
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| CN112574927A (en) * | 2020-12-31 | 2021-03-30 | 广西大学 | Selenium-resistant strain GX-B4 and application thereof |
| CN113322212A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-1 strain Dyella sp and application thereof in oxidizing organic selenium or elemental selenium |
| CN113322211A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof |
| CN113430136A (en) * | 2021-07-09 | 2021-09-24 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113322212B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | LX-1 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113322211B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof |
| CN113430136B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN115820467B (en) * | 2022-09-23 | 2023-08-25 | 广西壮族自治区农业科学院 | Bacillus pumilus TXB1-8 and application thereof |
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