CN105132314A - Brevibacterium frigoritolerans YLX-3 and application thereof - Google Patents
Brevibacterium frigoritolerans YLX-3 and application thereof Download PDFInfo
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Abstract
The invention discloses brevibacterium frigoritolerans YLX-3 separated from a microbial mat sediment in a percolating water system in the selenium mining area of Enshi, Hubei, China and application of brevibacterium frigoritolerans YLX-3. The brevibacterium frigoritolerans YLX-3 is preserved in CCTCC (China Center for Type Culture Collection) with the preservation number of CCTCC No: M2013673. The brevibacterium frigoritolerans YLX-3 has the characteristics of selenium-addicted microbes and has a potential application value in microbial organic selenium conversion, microbial nano-selenium synthesis, selenium-containing ore microbial activation, selenium-rich microbial fertilizer preparation and selenium-contaminated environment remediation.
Description
Technical field
The present invention relates to a kind of addicted to selenium microorganism and application thereof, particularly relate to a strain brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 and the application in microbial transformation synthesis organoselenium, Microbe synthesis nanometer selenium, activation of microorganism selenium ore, selenium-enriched microbe fertilizer and selenium contamination environment remediation thereof, belong to biological technical field.
Background technology
Extreme microorganism refers to the microorganism kind being adapted at living in extreme environment, comprise microorganism (Rothschild & Mancinelli, 2001 of thermophile bacteria, halophilic bacterium, basophilic bacterium, acidophilic bacteria, barophilic bacteria, psychrophilic bacteria and radioprotective, resistance to drying, anti-high-concentration metallic ions and extreme anaerobism; Chen Jun etc., 2006), the research of current extreme microorganism is mainly in regions such as mid-ocean rise, hot spring, salt lake, mine drainages.To live in the hydrothermal solution environment of deep-sea mid-ocean ridge thermophilic, barophilic bacteria in a large number, its Fe, S, Zn, Cu at mid-ocean ridge circulation and become in ore deposit to play important role (Taylor & Wirsen, 1997; Labrenzetal., 2000; Kasama & Murakami, 2001; Emerson & Mayer, 2002; Kennedyetal., 2003; Peng Xiaotong etc., 2007; Chen Shun etc., 2010).Geomicrobiology scholar is to U.S. Yellowstone (Susanetal., 1994,1996), Iceland (Marteinssonetal., 2001; Thomasetal., 2007), Italy (Kvistetal., 2005), Chinese yunnan (Song Zhaoqi etc., 2008; Huang Qiuyuan etc., 2010) etc. numerous hot springs on ground are studied, and are disclosed in these up to more than 70 degrees Celsius, inside some hot spring even more than 100 degrees Celsius, enliven a large amount of thermophilic microorganism---the ancient bacterium of spring, the imagination to the microbe survival limit before having expanded us.And Utah,U.S.A Great Salt lake (salinity 2.2%), the Dead Sea (salinity 2.5%), the Caspian Sea (live in the hypersaline environment such as salinity (1.7%) many anti-high osmotic pressure microorganisms (Antonioetal., 1998; Vreelandetal., 1998; Orenetal., 2001).Be positioned at the maximum land plateau salt water lake of northeast of Qinghai province within Chinese territory, more due to the sedimentary environment of its uniqueness, become the natural laboratory (appropriate enter just etc., 2005) of geomicrobiology research.Exist addicted to sour iron-oxidizing bacteria in a large number in acid mine drainage, as thiobacillus ferrooxidant (Acidithiobacillusferrooxidans), participate in circulation (Baker & Banfield, 2003 of Fe, Mn, S etc. in a large number; Fortin & Langley, 2005; Lu Jianjun etc., 2005; Jiang Lei etc., 2006), cause ecological problem widely on the one hand, on the other hand for the improvement of mine drainage provides new thinking (Johnson & Kevin, 2003).Even if for the subglacial lake Living system of sealing millions of year under the thick ice sheet in the remote South Pole up for safekeeping, we are in store keen interest (Bulatetal., 2009,2011) still.
, few people pay close attention to the microorganism composition in rich selenium extreme environment.This is mainly because selenium is trace element in the earth's crust, is difficult to into ore deposit or forms the environment of extreme rich selenium, strongly limit the research addicted to selenium microorganism.
Enshi fishing dyke has Se fractionation unique in the world, typical case and unique extreme rich selenium environment, there is in the water from diffusion system of the water from diffusion system in selenium mining area and selenium ore deposit mine tailing the selenium content of high density, selenium content wherein in water body is up to 40.0-94.1 μ g/L, mean value is 58.4 ± 16.8 μ g/L (Zhu & Zheng, 2001; Zhuetal., 2008), being 330 times (Fordyceetal., 2000) of Area of Keshan Disease water body selenium content, is 10 times (Presser, 1994) that the tap water selenium content upper limit is recommended by the World Health Organization (WHO); Selenium content in settling is up to 8.28-82.9mg/kgDW, and mean value is 26.6 ± 26.8mg/kgDW (Zhu & Zheng, 2001; Zhuetal., 2008), be 19 times (Presser, 1994) of Western United States's settling selenium content higher limit (1.4mg/kgDW).At present, Jin You Zhu Jianming group has carried out preliminary study (Lei Lei etc. to the Microorganism diversity profiles in enshi fishing dyke height selenium carbonaceous mudstone,, and be separated the bacterium (Wang Mingyi etc., 2006) that two strains have selenite reducing power 2009).In patent of invention, only have in " a kind of method utilizing fermentable to produce activation selenium mineral powder " (ZL201210166664.4) and " a kind of utilize super resistance to selenium microorganism to prepare the method for red elemental selenium " (application number 201210167650.6) utilize the selenium slag of enshi selenium minerals, soil carries out being separated resistance to selenium microorganism of being correlated with, and then be inoculated into selenium mineral powder or other are containing in selenium medium, preparation activation selenium mineral powder or red elemental selenium.But separation and purification is not correlated with resistance to selenium microorganism, non-Identifying micro-organisms kind and feature yet in patent of invention.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3.
Technical scheme of the present invention is:
The invention provides a strain brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3, this oxidation microbacterium is separation and purification from the bacterium seat settling of selenium mining area, Chinese enshi water from diffusion system, there is the bacterium new strains addicted to selenium microorganism feature, the preservation name of this brevibacterium frigoritolerans is called brevibacterium frigoritolerans YLX-3 (BrevibacteriumfrigoritoleransYLX-3), depositary institution is China typical culture collection center (CCTCC), preservation address is: Wuhan, China Wuhan University, preservation date is on December 18th, 2013, deposit number is CCTCCNO:M2013673.
The bacterial classification mycology feature description of this brevibacterium frigoritolerans is as follows:
It is yellow level and smooth circle that [morphological specificity] this brevibacterium frigoritolerans cultivates the bacterium colony observed for 24 hours in the TSA substratum of 28 degrees Celsius, and has full edge convex surface (accompanying drawing 1); Gramstaining is carried out to bacterial strain, is shown as gram-positive microorganism.This bacterial strain is rod-short, and size is 0.8 ~ 1.0 × 1.0 ~ 2.0 μm (accompanying drawings 2);
[addicted to selenium feature] this brevibacterium frigoritolerans is linked into successively respectively containing selenium (with Sodium Selenite configuration) 0,5,10,100,300,500,1000, in the TSB nutrient solution of 5000mg/L, result shows: in selenium concentration≤5mg/L situation, bacterial growth is not suppressed, and nutrient solution color does not present obvious redness.In selenium concentration >=10mg/L situation, nutrient solution color starts to present obvious redness, and under 300mg/L condition, the redness in nutrient solution is still obvious, shows this bacterial classification Metabolism of Normal.Even if it must be noted that, under the extreme condition of selenium content up to 1000mg/L, bacterial classification still can grow, embodied extreme Selenium endurance, but nutrient solution redness obviously weakens.More than under 1000mg/L concentration, this bacterial classification can not grow.
[16SrRNA gene sequencing] extracts genomic dna from the pure growth of bacterial strain of the present invention, universal primer 27f and 1492r is utilized to carry out pcr amplification and order-checking, further by CLUSTALX software and Mega4.0 software with Neighbour-joining method constructing system evolutionary tree (accompanying drawing 3), carry out Phylogenetic Analysis.Result display bacterial strain belongs to brevibacterium frigoritolerans and belongs to (Brevibacteriumfrigoritolerans), called after brevibacterium frigoritolerans YLX-3 (BrevibacteriumfrigoritoleransYLX-3), depositary institution is China typical culture collection center (CCTCC), preservation address is: Wuhan, China Wuhan University, preservation date is on December 18th, 2013, and deposit number is CCTCCNO:M2013673.
The enzyme of the API20NE (non-bowel gram negative bacillus identification systems) of [biochemical reactions feature] this bacterial strain and carbon source through fermentation reaction and display, bacterial strain can react with arginine dihydrolase, EC 1.4.1.19, and can react with 3-Hydroxybutanone and produce acetyl methyl carbinol.Bacterial strain can utilize N.F,USP MANNITOL and amygdaloside as carbon source through fermentation (table 1) well.This strain growth characteristic and Enzyme activity assay display, bacterial strain 2% and 5%NaCl growth from solution good, the esterase (C of bacterial strain
4), lipoid esterase (C
8), naphthols-AS-BI-phosphohydrolase, β-gala neuraminidase, alpha-glucosidase, beta-glucosidase all have stronger activity (table 2).
The enzyme of table 1API20NE and carbon source through fermentation
| Numbering | The enzyme measured and physiological property detect | Result |
| 1 | Beta-galactosidase enzymes | - |
| 2 | Arginine dihydrolase | + |
| 3 | Lysine decarboxylase | - |
| 4 | Ornithine decarboxylase | - |
| 5 | Citric acid utilizes | - |
| 6 | H 2S produces | - |
| 7 | Urase | - |
| 8 | Tryptophane desaminase | + |
| 9 | Indoles produces | - |
| 10 | 3-Hydroxybutanone produces acetyl methyl carbinol | + |
| 11 | Gelatinase | - |
| 12 | Glucose fermentation/oxidation | - |
| 13 | Mannose ferment/oxidation | + |
| 14 | Inositol fermentation/oxidation | - |
| 15 | Sorbitol fermentation/oxidation | - |
| 16 | Rhamnosyl fermentation/oxidation | - |
| 17 | Sucrose fermentation/oxidation | - |
| 18 | Melibiose fermentation/oxidation | - |
| 19 | Amygdaloside fermentation/oxidation | + |
| 20 | Pectinose fermentation/oxidation | - |
Note: "+" represents positive, "-" represents negative, and " v " represents the weak positive.
Table 2 strain growth characteristic and Enzyme assay
| Numbering | The enzyme measured and physiological property detect | Result |
| 1 | 2%, 5%NaCl concentration growing state | + |
| 2 | Amylase | - |
| 3 | Alkaline phosphate enzyme | - |
| 4 | Esterase (C 4) | + |
| 5 | Esterase lipase (C 8) | + |
| 6 | Lipase (C 14) | - |
| 7 | L-LEU arylamine enzyme | - |
| 8 | α-amino-isovaleric acid arylamine enzyme | - |
| 9 | Gelucystine arylamine enzyme | - |
| 10 | Trypsinase | - |
| 11 | Quimotrase | - |
| 12 | Acid phosphatase | - |
| 13 | Naphthols-AS-BI-phosphohydrolase | + |
| 14 | Alpha-galactosidase | - |
| 15 | Beta galactoside enzyme | + |
| 16 | β-uronic acid glucoside enzyme | - |
| 17 | Alpha-gluconase activity enzyme | + |
| 18 | β-glucosaccharase | + |
| 19 | N-acetyl-glucosaminidase | - |
| 20 | α-mannosidase | - |
| 21 | β-Fucose glucoside enzyme | - |
Note: "+" represents positive, "-" represents negative.
The invention also discloses the application of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 at preparation microbial transformation organoselenium.Brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microorganism feature, the soluble state inorganic selenium (Se in nutrient solution
4+, Se
6+) under content is no more than the condition of 5mg/L, can by inorganic selenium Efficient Conversion for containing selenoamino acid (as selenocystine SeCys
2), these organoselenium can be utilized further to originate as the benefit selenium of selenium deficiency crowd.
The invention also discloses the application of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 in preparation microbial transformation nanometer selenium.Brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microorganism feature, the soluble state inorganic selenium (Se in nutrient solution
4+, Se
6+) content is under the condition of 10mg/L ~ 300mg/L, can be nanometer selenium by inorganic selenium Efficient Conversion, this nanometer selenium be of a size of 100 ~ 200nm, these can be utilized further to have bioactive nanometer selenium and originate as the benefit selenium of selenium deficiency crowd.
The invention also discloses brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 in activation containing the application in selenium ore.Brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microorganism feature, activation of microorganism can be carried out to containing selenium ore, produce free state selenium/biological nano selenium, and then make containing in selenium ore can not the activation of biological utilisation selenium being bioavailable selenium, 8 ~ 10 times can be improved containing bioavailable selenium content in selenium ore by natural, further can by activation after containing selenium ore processing be Selenium in Soil modifying agent, be applied in scarce selenium soil and plant selenium-enriched crop.
The invention also discloses brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 and prepare the application in selenium-enriched microbe fertilizer.Brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microorganism feature, can directly be made as microorganism lyophilized powder, be administered to rehydration after in soil to bring back to life, selenium in activating soil, improve the bioavailability of Selenium in Soil, thus improve crop to the cumulative efficiency of selenium; Also can with fertilizer complex ferment, make microbial organic fertilizer, be applied in soil and plant selenium-enriched crop.
The invention also discloses the application of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 in selenium contamination environment remediation.Brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microorganism feature, when selenium content in polluted-water is no more than 100 μ g/L, microbial transformation can be carried out to the selenium in selenium contamination environment, accumulation, these microorganisms can be collected further, greatly reduce the selenium content in environment on the one hand, reach the object of repairing environment, conversion can be utilized on the other hand, the microorganism selenium of accumulation, as the source of microorganism organoselenium or microorganism nanometer selenium, selenium for scarce selenium crowd supplements source, it can reach 80% to the elimination efficiency of selenium in selenium contamination water body.
By such scheme, the present invention at least has the following advantages: brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 of the present invention has addicted to selenium microbiological property, can transform at microorganism organoselenium, microorganism nanometer selenium synthesis, containing in selenium ore activation of microorganism, selenium-enriched microbe fertilizer and selenium contamination environment remediation, there is potential using value.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the morphological specificity of bacterial strain of the present invention;
Fig. 2 is bacterial strain of the present invention morphological specificity under the microscope;
Fig. 3 is the phylogenetic tree schematic diagram of the 16SrRNA gene order of bacterial strain of the present invention.
Embodiment
Below in conjunction with drawings and Examples, be described in further detail the specific embodiment of the present invention, following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Bacteria selection separation is carried out according to following step:
(1) primary dcreening operation: the bacterium seat sediment sample that aseptic collection Hubei China is bestowed favour in the water from diffusion system of fishing dyke selenium mining area is about 20g in the sealed bottle of the 25mL of sterilising treatment, parallel acquisition 3 parts, takes back 4 DEG C, laboratory cryopreservation.Then 3 parts of parallel sample mixed in the aseptic operating platform of laboratory, get the Erlenmeyer flask that 5g is placed in 50mL sterile purified water, under normal temperature condition, 150r/min shakes 15min, leaves standstill 30 seconds Hou Qu1mLJun liquid echelons and is diluted to 10
-3, 10
-4, 10
-5, 10
-6, then inoculate 0.05mL and be spread evenly across (isolation medium forms: containing the TSA of selenium 200 μ g/kg, selenium source is Sodium Selenite) on isolation medium, each process arranges 4 groups of repetitions.Cultivate 2-3 days under normal temperature, the typical single bacterium colony of picking form carries out point and connects purifying and obtain pure growth 2-3 time.
(2) multiple sieve: the pure growth obtained in (1) is accessed successively containing selenium (configuring with Sodium Selenite) 0,5ppm, 10,100,300,500,1000, in the TSB nutrient solution of 5000mg/kg, under selenium concentration≤1000mg/kg condition, this bacterial strain is energy normal growth all, show that this pure growth has certain Selenium endurance, and called after YLX-3, be preserved in storage medium.
The brevibacterium frigoritolerans obtained is sieved again to above-mentioned warp and carries out morphological specificity observation, molecular biology identification and biochemical reactions characteristic test.
Morphological specificity is observed: it is yellow level and smooth circle that this brevibacterium frigoritolerans cultivates the bacterium colony observed for 24 hours in the TSA substratum of 28 degrees Celsius, and has full edge convex surface (shown in accompanying drawing 1); Gramstaining is carried out to bacterial strain, is shown as gram-positive microorganism.Its bacterial strain observed under transmission electron microscope is rod-short, and size is 0.8 ~ 1.0 × 1.0 ~ 2.0 μm (accompanying drawings 2);
Molecular biology identification: extract genomic dna from the pure growth of bacterial strain of the present invention, universal primer 27f and 1492r is utilized to carry out pcr amplification and order-checking, further by CLUSTALX software and Mega4.0 software with Neighbour-joining method constructing system evolutionary tree (accompanying drawing 3), carry out Phylogenetic Analysis.Result display bacterial strain belongs to brevibacterium frigoritolerans and belongs to (Brevibacteriumfrigoritolerans), called after brevibacterium frigoritolerans YLX-3 (BrevibacteriumfrigoritoleransYLX-3), depositary institution is China typical culture collection center (CCTCC), preservation address is: Wuhan, China Wuhan University, preservation date is on December 18th, 2013, and deposit number is CCTCCNO:M2013673.
Biochemical reactions characteristic test: the enzyme of the API20NE (non-bowel gram negative bacillus identification systems) of this bacterial strain and carbon source through fermentation reaction and display, bacterial strain can react with arginine dihydrolase, EC 1.4.1.19, and can react with 3-Hydroxybutanone and produce acetyl methyl carbinol.Bacterial strain can utilize N.F,USP MANNITOL and amygdaloside as carbon source through fermentation (table 1) well.This strain growth characteristic and Enzyme activity assay display, bacterial strain 2% and 5%NaCl growth from solution good, the esterase (C of bacterial strain
4), lipoid esterase (C
8), naphthols-AS-BI-phosphohydrolase, β-gala neuraminidase, alpha-glucosidase, beta-glucosidase all have stronger activity (table 2).
Embodiment 1
The application of microorganism of the present invention in synthesis organoselenium.
By microorganism YLX-3 inoculation of the present invention in the TSB nutrient solution containing selenium (Sodium Selenite configuration) 1.5mg/L, under normal temperature, 200r/min cultivates 48 hours, collected by centrifugation thalline and supernatant liquor.Then utilize liquid chromatography-atomic fluorescence spectroscopy (AFS) combined instrument (LC-GA-AFS) to detect selenium content in supernatant liquor and thalline and form respectively, in result display supernatant liquor, be almost Se
4+, and in thalline, be selenocystine (SeCys
2) (table 4).This shows that YLX-3 bacterial strain can under 1.5mgSe/L condition, by the inorganic selenium (Se in nutrient solution
4+) be converted into organoselenium (SeCys
2), and be accumulated in thalline.The organoselenium that further collection thalline can be used as animal-feed etc. adds, and also can be further seleno-amino acids purified feed stock (especially selenocystine SeCys
2), the organoselenium as scarce selenium crowd mends selenium source.
The Se form feature of supernatant liquor and thalline in table 4 reaction system
Note: "-" expression does not detect; " * " represents do not have sample.
Embodiment 2
The application of microorganism of the present invention in synthesis organoselenium.
By microorganism YLX-3 inoculation of the present invention in the TSB nutrient solution containing selenium (Sodium Selenite configuration) 5mg/L, under normal temperature, 200r/min cultivates 72 hours, collected by centrifugation thalline and supernatant liquor.Then utilize liquid chromatography-atomic fluorescence spectroscopy (AFS) combined instrument (LC-GA-AFS) to detect selenium content in supernatant liquor and thalline and form respectively, in result display supernatant liquor, be almost Se
4+, and in thalline, the overwhelming majority is selenocystine (SeCys
2), there are small part Se-Methylselenocysteine (SeMeCys) and Se in the growth stage of stable development of bacterial strain
4+(table 5).This shows that YLX-3 bacterial strain can under 5mgSe/L condition, by the inorganic selenium (Se in nutrient solution
4+) be converted into organoselenium (SeCys
2, SeMeCys), and to be accumulated in thalline.The organoselenium that further collection thalline can be used as animal-feed etc. adds, and also can be further seleno-amino acids purified feed stock (especially selenocystine SeCys
2with methyl selenocystine SeMeCys), the organoselenium as scarce selenium crowd mends selenium source.
The Se form feature of supernatant liquor and thalline in table 5 reaction system
Note: "-" expression does not detect; " * " represents do not have sample.
Embodiment 3
The application of microorganism of the present invention in synthesis of nano selenium.
By microorganism YLX-3 inoculation of the present invention in the TSB nutrient solution containing selenium (Sodium Selenite configuration) 10mg/L, under normal temperature, 200r/min cultivates 24 hours, and solution occurs significantly red, muddy.Cultivate 48 hours, collected by centrifugation thalline and supernatant liquor, what discovery collection obtained is precipitated as red suspended substance.Then utilize liquid chromatography-atomic fluorescence spectroscopy (AFS) combined instrument (LC-GA-AFS) and Atomic Fluorescence Spectroscopy (AFS), the composition of red suspended substance is almost elemental selenium to utilize minusing to identify.The nanometer selenium of this Microbe synthesis can be originated as the benefit selenium of scarce selenium crowd, also can as selenium fertilizer or animal feedstuff additive.
Embodiment 4
The application of microorganism of the present invention in synthesis of nano selenium.
By microorganism YLX-3 inoculation of the present invention in the TSB nutrient solution containing selenium (Sodium Selenite configuration) 300mg/L, under normal temperature, 200r/min cultivates 12 hours, and solution occurs significantly red, muddy.Cultivate 48 hours, collected by centrifugation thalline and supernatant liquor, what discovery collection obtained is precipitated as red suspended substance.Then utilize liquid chromatography-atomic fluorescence spectroscopy (AFS) combined instrument (LC-GA-AFS) and Atomic Fluorescence Spectroscopy (AFS), the composition of red suspended substance is almost elemental selenium to utilize minusing to identify.The nanometer selenium of this Microbe synthesis can be originated as the benefit selenium of scarce selenium crowd, also can as selenium fertilizer or animal feedstuff additive.
Embodiment 5
The application of microorganism of the present invention in activation selenium ore.
To the selenium ore grinding in selenium mining area, enshi be picked up to 100 orders, then measure its total selenium content and be about 3000mg/kg, further by continuous extraction method, the water soluble speciation measured in selenium ore is about accounting 1%, exchangeable species selenium is about accounting 3%, Fe-Mn oxide selenium is about accounting 5%, organically combines state selenium and is about accounting 10%, residual selenium accounting about 81%.With this selenium mineral powder for selenium source, add this selenium mineral powder of 100g in the TSB nutrient solution of 300mL, do not inoculate YLX-3 (8 Duplicate Samples) for one group, another group inoculation YLX-3 (8 Duplicate Samples).200r/min cultivates 90 days at ambient temperature, and the total selenium content (table 6) detected according to certain time interval sampling in supernatant liquor, in result display inoculation YLX-3 bacterial strain group, obviously comparatively control group has more soluble state selenium to enter in solution, and its selenium content is about 10 times of control group.This shows that YLX-3 bacterial strain can not be converted into bioavailable selenium by biological utilisation selenium (as exchange state selenium, Fe-Mn oxide selenium) by selenium ore, and efficiency is higher.This characteristic of YLX-3 can be utilized further to activate natural selenium ore, and product can be used as Selenium in Soil modifying agent, for the improvement of scarce selenium soil.
Selenium content feature in table 6 reaction system supernatant liquor
Embodiment 6
The application of microorganism of the present invention in the preparation of selenium-enriched microbe fertilizer.
Microorganism YLX-3 bacterial strain of the present invention is carried out 50L mass-producing fermentation culture 3-5 days in TSB nutrient solution, adopts freeze drying technology to prepare microorganism lyophilized powder further.This lyophilized powder can be applied directly in romaine lettuce crop root soil, waters subsequently and freeze-dried microorganisms rehydration can be made to bring back to life.Normal cultivation was gathered in the crops after 30 days, and the selenium content in its Chinese leaf of detection display is 2-3 times of the Chinese leaf sample not using this microorganism lyophilized powder.
Embodiment 7
The application of microorganism of the present invention in the preparation of selenium-enriched microbe fertilizer.
By microorganism YLX-3 inoculation of the present invention in organic fertilizer raw material, carry out normal fertilizer compost fermentation subsequently, obtain microbial organic fertilizer.This microbial organic fertilizer is administered to romaine lettuce root, and normal cultivation after 30 days is gathered in the crops, and the selenium content in its Chinese leaf of detection display is 2-3 times of the Chinese leaf sample not using this microorganism lyophilized powder.
Embodiment 8
The application of microorganism of the present invention in selenium contamination environment remediation.
Measure total selenium content in the water from diffusion of selenium mining area, enshi and be about 85 μ g/L, and it can be used as the representative of selenium contamination environment water, by in this water body of YLX-3 bacterial classification access 300mL (8 Duplicate Samples), and add glucose as carbon source, control group is set simultaneously, 200r/min cultivates 10 days at ambient temperature, and the total selenium content (table 7) detected according to certain time interval sampling in supernatant liquor, it is substantially constant that result shows the selenium content do not inoculated in the control group of YLX-3 bacterial strain in solution, and inoculate YLX-3 bacterial strain treatment group in strain growth process obviously by the selenium absorption in solution, make use of, greatly reduce the selenium content in solution, clearance rate reaches about 80%.Therefore, this characteristic of YLX-3 can be utilized further to carry out repair process to the water body in natural selenium contamination area (as bestowing favour) or artificial selenium contamination environment (as manganese smelts region).
Selenium content feature in table 7 reaction system supernatant liquor
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. a brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3, it is characterized in that: the preservation name of this brevibacterium frigoritolerans is called brevibacterium frigoritolerans YLX-3 (BrevibacteriumfrigoritoleransYLX-3), depositary institution is China typical culture collection center, preservation date is on December 18th, 2013, and deposit number is CCTCCNO:M2013673.
2. brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim 1, it is characterized in that: the bacterial strain of described brevibacterium frigoritolerans is rod-short, size is 0.8 ~ 1.0 × 1.0 ~ 2.0 μm, bacterium colony is yellow level and smooth circle, and there is full edge convex surface, Gram-positive.
3. brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim 1, is characterized in that: when soluble state selenium content is lower than 5mg/L, and this soluble state inorganic selenium sorption enhanced can be organoselenium by described brevibacterium frigoritolerans; When soluble state selenium content is 10 ~ 300mg/L, this soluble state inorganic selenium can be converted into by described brevibacterium frigoritolerans has bioactive nanometer selenium, and this nanometer selenium is of a size of 100 ~ 200nm.
4. the application of the brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim arbitrary in claims 1 to 3, is characterized in that: described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 is in the application of preparation microbial transformation organoselenium.
5. the application of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim 4, it is characterized in that: this soluble state inorganic selenium sorption enhanced, when soluble state selenium content is lower than 5mg/L, can be organoselenium by described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3.
6. the application of the brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim arbitrary in claims 1 to 3, is characterized in that: the application of described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 in preparation microbial transformation nanometer selenium.
7. the application of brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim 6, it is characterized in that: this soluble state inorganic selenium, when soluble state selenium content is 10 ~ 300mg/L, can be converted into and has bioactive nanometer selenium by described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3.
8. the application of the brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim arbitrary in claims 1 to 3, is characterized in that: described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 contains the application in selenium ore in activation.
9. the application of the brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim arbitrary in claims 1 to 3, is characterized in that: describedly preparing the application in selenium-enriched microbe fertilizer addicted to selenium microorganism.
10. the application of the brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 according to claim arbitrary in claims 1 to 3, is characterized in that: the application of described brevibacterium frigoritolerans BrevibacteriumfrigoritoleransYLX-3 in selenium contamination environment remediation.
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| CN105969705A (en) * | 2016-07-30 | 2016-09-28 | 袁志辉 | Stabilizing agent for selenium-contaminated soil repairing bacteria |
| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN109706096A (en) * | 2019-01-04 | 2019-05-03 | 广东博沃特生物科技有限公司 | One plant of brevibacterium frigoritolerans and its application with denitrogenation and efficient flocculating ability |
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| CN111979152A (en) * | 2020-08-25 | 2020-11-24 | 甘肃省科学院生物研究所 | Compound microbial agent for deodorizing livestock and poultry manure and preparation method thereof |
| CN113373094A (en) * | 2021-07-16 | 2021-09-10 | 榆林市中泰农业科技有限公司 | Brevibacterium fritolerans SDB5 and application thereof in promoting plant growth |
| CN113373094B (en) * | 2021-07-16 | 2022-02-01 | 榆林市中泰农业科技有限公司 | Brevibacterium fritolerans SDB5 and application thereof in promoting plant growth |
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