WO2017035856A1 - 嗜硒微生物Wautersiella enshiensis YLX-1及其应用 - Google Patents

嗜硒微生物Wautersiella enshiensis YLX-1及其应用 Download PDF

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WO2017035856A1
WO2017035856A1 PCT/CN2015/089320 CN2015089320W WO2017035856A1 WO 2017035856 A1 WO2017035856 A1 WO 2017035856A1 CN 2015089320 W CN2015089320 W CN 2015089320W WO 2017035856 A1 WO2017035856 A1 WO 2017035856A1
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selenium
microorganism
ylx
enshiensis
wautersiella
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French (fr)
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袁林喜
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苏州硒谷科技有限公司
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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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Definitions

  • the invention relates to a selenium-secreting microorganism and application thereof, in particular to a selenium-preserving microorganism Wautesiella enshiensis YLX-1 and its preparation for microbial conversion to organic selenium, microbial synthesis of nano selenium, microbial activated selenium ore, selenium-enriched microbial fertilizer and selenium
  • the application in the restoration of polluted environment belongs to the field of biotechnology.
  • Extreme microorganisms are microbial species suitable for living in extreme environments, including thermophiles, halophilic bacteria, alkalophils, acidophilic bacteria, pressure-sensitive bacteria, psychrophilic bacteria, and radiation-resistant, dry-resistant, high-resistance metals. Ions and extremely anaerobic microorganisms (Rothschild & Mancinelli, 2001; Chen Jun et al., 2006), the current research on extreme microorganisms is mainly in the mid-ocean ridge, hot springs, salt lakes, mine drainage and other areas.
  • thermophilic and pressure-sensitive bacteria in the hydrothermal environment of the deep ocean ridge, which plays an important role in the Fe, S, Zn, Cu cycle and mineralization of the mid-ocean ridge (Taylor & Wirsen, 1997; Labrenz et Al., 2000; Kasama & Murakami, 2001; Emerson & Mayer, 2002; Kennedy et al., 2003; Peng Xiaotong et al., 2007; Chen Shun et al., 2010).
  • Hubei Enshi Yutangba has the world's only selenium deposit, which is a typical and unique extreme selenium-enriched environment. It has a high concentration of selenium in the percolating water system of selenium ore and the percolating water system of selenium mine tailings.
  • the selenium content is as high as 40.0-94.1 ⁇ g/L, with an average of 58.4 ⁇ 16.8 ⁇ g/L (Zhu & Zheng, 2001; Zhu et al., 2008), which is 330 times the selenium content of water in Keshan disease area (Fordyce et al.
  • the present invention provides a selenium-preserving microorganism, Wautersiella enshiensis YLX-1, and an application thereof.
  • the invention provides a selenium-preserving microorganism, Wautersiella enshiensis YLX-1, which is a new bacterial species with selenium-trapping microbial characteristics which is separated and purified from the bacterial sediment of the percolating water system of the Enshi selenium mining area in Hubei, China.
  • the selenium-enriched microorganism is deposited under the name of Warthella YLX-1 (Wautersiella enshiensis YLX-1), and the deposit is the China Center for Type Culture Collection (CCTCC).
  • the deposit address is: Wuhan University, Wuhan, China, and the deposit date is 2013. On the 18th of the month, the deposit number is CCTCCNO: M2013671.
  • the strains of the present invention are sequentially introduced into a TSB culture solution containing selenium (sodium selenite) at 0, 10, 100, 500, 1000, 3000, 6000 mg/L, and the results are shown.
  • selenium sodium selenite
  • bacterial growth was not inhibited (Fig. 3), and the color of the culture solution did not show a distinct red color (Fig. 5).
  • selenium concentration ⁇ 100mg/L
  • the growth of the strain in the 100mg/L selenium-containing medium was the fastest, 500mg/L, but the growth was slowed down in 1000mg/L, 3000mg/L and 6000mg/L.
  • Genomic DNA was extracted from the pure culture of the strain of the present invention, and PCR amplification and sequencing were carried out by using universal primers 27f and 1492r, and further constructed by the Neighbour-joining method by CLUSTAL X software and Mega 4.0 software.
  • Phylogenetic tree ( Figure 6) for phylogenetic analysis. The results showed that the strain belonged to the genus of the genus Wautesi, but the closest similarity of the genus Wautesiella falsenii NF 993 T was 96.7%. Combined with its morphological characteristics, it was identified as a new species of Wardella, named Wautesiella enshiensis YLX. -1, deposited at the China Center for Type Culture Collection (CCTCC) on December 18, 2013, and deposited at Wuhan University, Wuhan, China, with accession number: CCTCC No: M2013671.
  • the enzyme ZYM (enzyme activity) assay of the strain showed alkaline phosphatase, esterase (C 4 ), lipoesterase (C 8 ), white (leucine) arylase, trypsin, Acid phosphatase, naphthol-AS-BI-phosphohydrolase, ⁇ -glucosidase, and ⁇ -glucosidase all have strong activity, and proline arylamine has weak activity (Table 2).
  • the growth temperature of the strain is 4-37 ° C (optimal temperature is 20 ° C), pH is 5.0-9.0 (optimal pH is 7), salinity is 0-5% (more than 5% does not grow), the strain is Facultative anaerobic bacteria with no motility and hydrolyzable Tween 40 (Table 3).
  • the DNA G+C molar ratio of the strain was 31.6%
  • the main respiratory sputum was MK-6
  • the main fatty acids were C 16:1 ⁇ 6c, C 16:1 ⁇ 7c, iso-C 15:0 , iso-C 17:0 3OH, C 16:0 , C 16:1 ⁇ 5c, iso-C 17:1 I/anteiso-C 17:1 B (Table 4).
  • the invention also discloses the application of the selenium-preserving microorganism Wautersiella enshiensis YLX-1 in preparing microorganisms for converting organic selenium.
  • the microorganism of the present invention Wautersiella enshiensis YLX-1, has the characteristics of selenium-trapping microorganisms, and the inorganic selenium can be efficiently used under the condition that the soluble inorganic selenium (Se 4+ , Se 6+ ) content in the culture solution does not exceed 10 mg/L.
  • Conversion to selenium-containing amino acids (such as selenocystine SeCys 2 ) can further utilize these organic selenium as a source of selenium supplementation for people with selenium deficiency.
  • the invention also discloses the application of the selenium-trapping microorganism Wautesiella enshiensis YLX-1 in preparing microbial transformed nano selenium.
  • the microorganism of the present invention Wautersiella enshiensis YLX-1, has the characteristics of selenium-trapping microorganisms, and the content of soluble inorganic selenium (Se 4+ , Se 6+ ) in the culture solution can be 100 mg/L to 6000 mg/L.
  • Inorganic selenium is efficiently converted into nano-selenium.
  • the size of the nano-selenium is 100-200 nm, and these biologically active nano-selenium can be further utilized as a source of selenium supplement for the selenium-deficient population.
  • the invention also discloses the application of the selenium-enhancing microorganism Wautersiella enshiensis YLX-1 in activating selenium-containing ore.
  • the microorganism of the present invention Wautersiella enshiensis YLX-1, has the characteristics of selenium-trapping microorganisms, and can activate microbes for selenium-containing ores, thereby enabling non-bioavailable selenium in selenium-containing ores to be activated into bio-available selenium, which can be used in natural selenium-containing ores.
  • the bioavailable selenium content is increased by 5 to 10 times, and the activated selenium ore can be processed into a soil selenium improver, and the selenium-enriched crop can be planted in the selenium-deficient soil.
  • the invention also discloses the application of the selenium-preserving microorganism Wautersiella enshiensis YLX-1 in preparing a selenium-enriched microbial fertilizer.
  • the microorganism of the present invention Wautersiella enshiensis YLX-1, has the characteristics of selenium-secreting microorganisms, can be directly prepared as a microbial lyophilized powder, is re-activated after re-watering after being applied to the soil, activates selenium in the soil, and improves the bioavailability of selenium in the soil, thereby Improve the accumulation efficiency of selenium in crops; it can also be fermented with organic fertilizer to make microbial organic fertilizer, and applied to soil to grow selenium-enriched crops.
  • the invention also discloses the application of the selenium-preserving microorganism Wautersiella enshiensis YLX-1 in the environmental protection of selenium pollution.
  • the microorganism of the present invention Wautersiella enshiensis YLX-1, has the characteristics of selenium-trapping microorganisms.
  • the selenium content in the polluted water body does not exceed 100 ⁇ g/L, the microorganisms in the selenium-contaminated environment can be microbial transformed and accumulated, and the microorganisms can be further collected.
  • the transformed and accumulated microbial selenium can be used as a source of microbial organic selenium or microbial nano selenium for selenium supplementation sources of selenium-deficient people. Its removal efficiency of selenium in selenium-contaminated water can reach 70-80%.
  • the present invention has at least the following advantages: the selenium-producing microbial strain of the present invention has selenium-containing microbial characteristics, and can be used in microbial organic selenium conversion, microbial nano-selenium synthesis, selenium-containing ore microbial activation, and selenium-enriched microbial fertilizer preparation. And the potential application value of selenium pollution environment restoration.
  • Figure 1 is a morphological feature of the strain of the present invention
  • Figure 2 is a morphological feature of the strain of the present invention under a microscope
  • Figure 3 is a graph showing growth characteristics of strains of the present invention in 0 and 10 mg Se/L culture solutions
  • Figure 4 is a graph showing growth characteristics of strains of the present invention in culture media of 100, 500, 1000, 3000 and 6000 mg Se/L;
  • Figure 5 is a color characteristic of the system after the growth of the strains of the present invention in 0, 10, 100, 500, 1000, 3000 and 6000 mg Se/L in 24 hours;
  • Figure 6 is a schematic diagram showing the phylogenetic tree of the 16S rRNA gene sequence of the strain of the present invention.
  • Figure 7 is a scanning electron micrograph of the strain of the present invention grown in a 100 mg Se/L TSB medium for 48 hours;
  • Figure 8 is a spectrum analysis (SEM-EDS) diagram of the strain of the present invention after growth for 48 hours in a 100 mg Se/L TSB medium;
  • Figure 9 is a scanning electron micrograph of the strain of the present invention grown in a 6000 mg Se/L TSB medium for 48 hours;
  • Figure 10 is a spectrum analysis (SEM-EDS) of the strain of the present invention after growth for 48 hours in 6000 mg Se/L TSB broth.
  • the echelon is diluted to 10 -3 , 10 -4 , 10 -5 , 10 -6 , and then inoculated with 0.05mL evenly coated on the separation medium (separation medium composition: TSA containing selenium 200 ⁇ g/kg, selenium source is selenium Sodium), 4 sets of replicates were set for each treatment. Incubate at room temperature for 2-3 days, pick a single colony with typical morphology and perform 2-3 times purification to obtain a pure culture.
  • separation medium composition TSA containing selenium 200 ⁇ g/kg, selenium source is selenium Sodium
  • Genomic DNA was extracted from the pure culture of the strain of the present invention, PCR amplification and sequencing were carried out by using universal primers 27f and 1492r, and phylogenetic evolution was further carried out by the Neighbour-joining method by CLUSTAL X software and Mega 4.0 software. Tree ( Figure 6) for phylogenetic analysis. The results showed that the strain belonged to the genus of the genus Wautesi, but the closest similarity of the genus Wautesiella falsenii NF 993 T was 96.7%. Combined with its morphological characteristics, it was identified as a new species of Wardella, named Wautesiella enshiensis YLX. -1, deposited at the China Center for Type Culture Collection (CCTCC) on December 18, 2013, and deposited at Wuhan University, Wuhan, China, with accession number: CCTCC No: M2013671.
  • CTCC China Center for Type Culture Collection
  • Physiological and biochemical reaction characteristic test The reaction of the API 20NE (non-intestinal Gram-negative bacilli identification system) of the strain of the present invention shows that the strain can well assimilate glucose, maltose, malic acid, sodium citrate, and can also be very It can hydrolyze esculetin ferric citrate and gelatin well and react with urea and p-nitro-D-methylgalactose (Table 1).
  • the enzyme ZYM (enzyme activity) assay of the strain showed alkaline phosphatase, esterase (C 4 ), lipoesterase (C 8 ), white (leucine) arylase, trypsin, Acid phosphatase, naphthol-AS-BI-phosphohydrolase, ⁇ -glucosidase, and ⁇ -glucosidase all have strong activity, and proline arylamine has weak activity (Table 2).
  • the growth temperature of the strain is 4-37 ° C (optimal temperature is 20 ° C), pH is 5.0-9.0 (optimal pH is 7), salinity is 0-5% (more than 5% does not grow), the strain is Facultative anaerobic bacteria with no motility and hydrolyzable Tween 40 (Table 3).
  • the DNA G+C molar ratio of the strain was 31.6%
  • the main respiratory sputum was MK-6
  • the main fatty acids were C 16:1 ⁇ 6c, C 16:1 ⁇ 7c, iso-C 15:0 , iso-C 17:0 3OH, C 16:0 , C 16:1 ⁇ 5c, iso-C 17:1 I/anteiso-C 17:1 B (Table 4).
  • the microorganism YLX-1 strain of the present invention was inoculated into a TSB culture solution containing selenium (sodium selenite) 1.5 mg/L, and cultured at 200 r/min for 48 hours at normal temperature, and the cells and the supernatant were collected by centrifugation. Then, the liquid chromatography-atomic fluorescence spectrometer (LC-GA-AFS) was used to detect the selenium content and morphology in the supernatant and the bacteria. The results showed that almost all of the supernatants were Se 4+ , and the cells were Most of them are selenocystine (SeCys 2 ) (Table 5).
  • selenium sodium selenite
  • the YLX-1 strain can convert inorganic selenium (Se 4+ ) in the culture solution into organic selenium (SeCys 2 ) under 1.5 mg Se/L and accumulate in the bacteria. Further collection of the bacteria can be used as organic selenium for animal feed, and can also be used as a further source of selenium amino acid purification as a source of selenium supplementation for selenium-deficient people.
  • the microorganism YLX-1 strain of the present invention was inoculated into a TSB culture solution containing selenium (sodium selenite) 10 mg/L, and cultured at 200 r/min for 72 hours at normal temperature, and the cells and the supernatant were collected by centrifugation. Then, the liquid chromatography-atomic fluorescence spectrometer (LC-GA-AFS) was used to detect the selenium content and morphology in the supernatant and the bacteria.
  • LC-GA-AFS liquid chromatography-atomic fluorescence spectrometer
  • the microorganism YLX-1 strain of the present invention was inoculated into a TSB culture solution containing selenium (sodium selenite) 100 mg/L, and cultured at 200 r/min for 24 hours at normal temperature, and the solution showed obvious redness and turbidity. After culturing for 48 hours, the cells and the supernatant were collected by centrifugation, and it was found that the collected precipitate was a red suspension. Then, using liquid chromatography-atomic fluorescence spectrometer (LC-GA-AFS) and atomic fluorescence spectrometer, the composition of the red suspension was identified by the subtractive method to be almost elemental selenium.
  • selenium sodium selenite
  • bio-available selenium can further be used as a source of selenium for animal feed to increase selenium levels in animals; it can also be used as a source of selenium supplement for people with selenium deficiency.
  • the microorganism YLX-1 strain of the present invention was inoculated into a TSB culture solution containing selenium (sodium selenite) 6000 mg/L, and cultured at 200 r/min for 12 hours at normal temperature, and the solution showed obvious redness and turbidity. After culturing for 48 hours, the cells and the supernatant were collected by centrifugation, and it was found that the collected precipitate was a red suspension. Then, using liquid chromatography-atomic fluorescence spectrometer (LC-GA-AFS) and atomic fluorescence spectrometer, the composition of the red suspension was identified by the subtractive method to be almost elemental selenium.
  • LC-GA-AFS liquid chromatography-atomic fluorescence spectrometer
  • microorganism of the invention in the activation of selenium ore.
  • the selenium ore collected from the Enshi Selenium Mine in Hubei province was pulverized to 100 mesh, and then the total selenium content was determined to be about 3000 mg/kg. Further, the continuous extraction method was used to determine the water-soluble selenium in the selenium ore, which accounted for about 1%, and the exchangeable state. Selenium accounts for about 3%, iron oxide and manganese oxide accounts for about 5%, organically bound selenium accounts for about 10%, and residual selenium accounts for about 81%.
  • this selenium ore powder as the selenium source, add 100g of the selenium ore powder to 300mL of TSB culture solution, one group is not inoculated with YLX-1 (8 parallel samples), and the other group is inoculated with YLX-1 (8 parallel samples) . Incubate at 200 r/min for 90 days at room temperature and sample the supernatant at regular intervals The total selenium content in the liquid (Table 7) showed that the inoculated YLX-1 strain group had significantly more soluble selenium into the solution than the control group, and its selenium content was 5-10 times that of the control group.
  • the YLX-1 strain can convert non-bioavailable selenium (such as exchangeable selenium, iron-manganese oxide selenium) in selenium ore into bioavailable selenium, and the efficiency is high. Further, this property of YLX-1 can be used to activate natural selenium ore, and the product can be used as a soil selenium improver for the improvement of selenium-deficient soil.
  • non-bioavailable selenium such as exchangeable selenium, iron-manganese oxide selenium
  • microorganism of the invention in the preparation of a selenium-enriched microbial fertilizer.
  • the microorganism YLX-1 strain of the present invention is subjected to 50L large-scale fermentation culture in TSB culture solution for 3-5 days, and the microorganism freeze-dried powder is further prepared by freeze-drying technique.
  • the lyophilized powder can be directly applied to the root soil of the lettuce crop, and then watering can revitalize the lyophilized microorganism. After 30 days of normal cultivation, the test showed that the selenium content in the lettuce leaves was 3-8 times that of the lettuce leaf samples to which the microorganism freeze-dried powder was not applied.
  • microorganism of the invention in the preparation of a selenium-enriched microbial fertilizer.
  • the microorganism YLX-1 strain of the present invention is inoculated into an organic fertilizer raw material, followed by normal organic fertilizer compost fermentation to obtain a microbial organic fertilizer.
  • the microbial organic fertilizer is applied to the root of the lettuce, and is harvested after 30 days of normal culture, and the test shows that the selenium content in the lettuce leaves is the lettuce which is not applied with the lyophilized powder of the microorganism.
  • Leaf samples are 3-5 times.
  • microorganism of the invention in the environmental repair of selenium pollution.
  • the total selenium content in the percolating water of the Enshi Selenium Mine in Hubei province was determined to be about 85 ⁇ g/L, and it was used as a representative of selenium-contaminated environmental water.
  • the YLX-1 strain was connected to 300 mL of the water (8 parallel samples) and glucose was added.
  • a control group was set at the same time, and cultured at 200 r/min for 10 days at room temperature, and the total selenium content in the supernatant was sampled at a certain time interval (Table 8), and the results showed that the YLX-1 strain was not inoculated.
  • the selenium content in the solution was basically unchanged, while the inoculated YLX-1 strain treatment group obviously absorbed and utilized the selenium in the solution during the growth process of the strain, which greatly reduced the selenium content in the solution, and the clearance rate reached 70- 80%. Therefore, this property of YLX-1 can be further utilized to repair water bodies in natural selenium-contaminated areas (eg, Enshi) or artificial selenium-contaminated environments (eg, manganese smelting areas).
  • natural selenium-contaminated areas eg, Enshi
  • artificial selenium-contaminated environments eg, manganese smelting areas

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Abstract

一种沃氏菌(Wautersiella enshiensis)菌株 YLX-1及其应用，该菌株保藏于中国武汉典型微生物保藏中心，保藏号为CCTCC No: M2013671。该菌株具有嗜硒菌特征，可用于有机硒的微生物转化、纳米硒的微生物合成、含硒矿石的微生物活化、富硒微生物肥料制备以及硒污染环境的修复。

Description

嗜硒微生物Wautersiella enshiensis YLX-1及其应用
本申请要求了申请日为2015年08月31日，申请号为201510547673.3，发明名称为“嗜硒微生物Wautersiella enshiensis YLX-1及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种嗜硒微生物及其应用，尤其涉及一种嗜硒微生物Wautersiella enshiensis YLX-1及其在微生物转化合成有机硒、微生物合成纳米硒、微生物活化硒矿石、富硒微生物肥料制备及硒污染环境修复中的应用，属于生物技术领域。
背景技术
极端微生物是指适合在极端环境中生活的微生物种属，包括嗜热菌、嗜盐菌、嗜碱菌、嗜酸菌、嗜压菌、嗜冷菌以及抗辐射、耐干燥、抗高浓度金属离子和极端厌氧的微生物(Rothschild&Mancinelli,2001；陈骏等，2006)，目前极端微生物的研究主要在大洋中脊、热泉、盐湖、矿坑排水等区域。在深海洋中脊的热液环境中生活着大量嗜热、嗜压细菌，其在洋中脊的Fe、S、Zn、Cu循环及成矿中扮演着重要的角色(Taylor&Wirsen,1997；Labrenz et al.,2000；Kasama&Murakami,2001；Emerson&Mayer,2002；Kennedy et al.,2003；彭晓彤等，2007；陈顺等，2010)。地质微生物学家对美国黄石公园(Susan et al.,1994,1996)、冰岛(Marteinsson et al.,2001；Thomas et al.,2007)、意大利(Kvist et al.,2005)、中国云南(宋兆齐等，2008；黄秋媛等，2010)等地的众多热泉进行研究，揭示在这些高达70摄氏度以上，有些甚至超过100摄氏度的热泉里面，活跃着大量嗜热微生物——泉古菌，拓展了我们之前对微生物生存极限的想象。而美国犹他大盐湖(盐度2.2％)、死海(盐度2.5％)、里海(盐度(1.7％)等高盐环境中生活着许多抗高渗透压微生物(Antonio et al.,1998；Vreeland et al.,1998；Oren et al.,2001)。位于青海省东北部的中国境内最大的内陆高原咸水湖泊，更由于其独特的沉积环境，成为地质微生物学研究的天然实验室(妥进才 等，2005)。酸性矿坑排水中大量存在嗜酸铁氧化菌，如氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)，大量参与Fe、Mn、S等的循环(Baker&Banfield,2003；Fortin&Langley,2005；陆建军等，2005；蒋磊等，2006)，一方面造成广泛的生态问题，另一方面为矿坑排水的治理提供了新的思路(Johnson&Kevin,2003)。即使对于遥远南极的厚厚冰层下封存几百万年之久的冰下湖生命系统，我们依然保存着浓厚的兴趣(Bulat et al.,2009,2011)。
可是，很少有人关注富硒极端环境中的微生物组成。这主要是因为硒是地壳中的微量元素，难以成矿或形成极端富硒的环境，极大限制了对嗜硒微生物的研究。
湖北恩施渔塘坝具有世界上唯一的硒矿床，是典型和独特的极端富硒环境，在硒矿区的渗滤水系统和硒矿尾矿的渗滤水系统中具有高浓度的硒含量，其中水体中的硒含量高达40.0-94.1μg/L，平均值为58.4±16.8μg/L(Zhu&Zheng,2001；Zhu et al.,2008)，是克山病区水体硒含量的330倍(Fordyce et al.,2000)，是世界卫生组织(WHO)推荐饮用水硒含量上限的10倍(Presser,1994)；沉积物中的硒含量高达8.28-82.9mg/kg DW，平均值为26.6±26.8mg/kg DW(Zhu&Zheng,2001；Zhu et al.,2008)，是美国西部沉积物硒含量上限值(1.4mg/kg DW)的19倍(Presser,1994)。目前，仅有朱建明小组对湖北恩施渔塘坝高硒碳质泥岩中的微生物多样性特征进行了初步研究(雷磊等，2009)，并分离了两株具有亚硒酸盐还原能力的细菌(王明义等，2006)。在发明专利方面，仅有《一种利用微生物发酵生产活化硒矿粉的方法》(ZL201210166664.4)和《一种利用超耐硒微生物制备红色单质硒的方法》(申请号201210167650.6)中利用湖北恩施硒矿物的硒矿渣、土壤进行分离相关耐硒微生物，进而接种到硒矿粉或其他含硒介质中，制备活化硒矿粉或红色单质硒。但发明专利中并未分离纯化相关耐硒微生物，也未鉴定微生物种属和特征。
发明内容
为解决上述技术问题，本发明提供了一种嗜硒微生物Wautersiella enshiensis YLX-1及其应用。
本发明的技术方案是：
本发明提供了一种嗜硒微生物Wautersiella enshiensis YLX-1，该嗜硒微生物是从中国湖北恩施硒矿区渗滤水系统的菌席沉积物中分离纯化的、具有嗜硒微生物特征的细菌新种，该嗜硒微生物的保藏名称为沃氏菌YLX-1(Wautersiella enshiensis YLX-1)，保藏单位为中国典型培养物保藏中心(CCTCC)，保藏地址为：中国武汉武汉大学，保藏日期为2013年12月18日，保藏编号为CCTCCNO:M2013671。
该嗜硒微生物的菌种菌学特征描述如下：
【形态特征】该嗜硒微生物在28摄氏度的TSA培养基中培养24小时观察到的菌落为黄色平滑的圆形，并具有全缘凸面(附图1)；对菌株进行革兰氏染色，显示为革兰氏阴性菌。透射电子显微镜下观察到的菌株为棒状、非孢子形成，大小为0.4～0.7×0.8～2.0μm(附图2)；
【嗜硒特征】将本发明所述菌种依次分别接入到含硒(以亚硒酸钠配置)0、10、100、500、1000、3000、6000mg/L的TSB培养液中，结果显示：在硒浓度≤10mg/L情况下，细菌生长不受抑制(附图3)，培养液颜色不呈现明显的红色(附图5)。在硒浓度≥100mg/L情况下，菌种在100mg/L含硒培养液中生长最快，500mg/L次之，但1000mg/L、3000mg/L和6000mg/L中生长依次明显减慢(附图4)，必须指出的是即使在硒含量高达6000mg/L的极端条件下，菌种依然能生长，体现出了极端耐硒能力。而且在硒浓度高于100mg/L条件下，培养液颜色明显呈现红色，那是因为细菌开始将亚硒酸盐转化为红色单质硒(附图5)。
【16S rRNA基因序列分析】从本发明所述菌株的纯培养物中提取基因组DNA，利用通用引物27f和1492r进行PCR扩增和测序，进一步通过CLUSTAL X软件和Mega 4.0软件以Neighbour-joining方法构建系统进化树(附图6)，进行系统发育分析。结果显示菌株属于沃氏菌属(Wautersiella)，但与其亲缘关系最近的Wautersiella falsenii NF 993T的序列相似性只有96.7％，结合其形态学特征，确定为沃氏菌新种，命名为Wautersiella enshiensis YLX-1，于2013年12月18日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉武汉大学， 保藏编号：CCTCC No:M2013671。
【生理生化反应特征】本发明所述菌株的API 20NE(非肠道革兰氏阴性杆菌鉴定系统)反应显示，菌株能很好地同化葡萄糖、麦芽糖、苹果酸、柠檬酸钠，也能很好地水解七叶灵柠檬酸铁、明胶，还能与脲素和对硝基-D-甲基半乳糖进行反应(表1)。该菌株的API ZYM(酶的活性)检测显示，菌株的碱性磷酸盐酶、酯酶(C4)、类脂酯酶(C8)、白(亮)氨酸芳氨酶、胰蛋白酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶、α-葡萄糖苷酶、β-葡萄糖苷酶均具有较强的活性，缬氨酸芳胺酶具有弱活性(表2)。该菌株的生长温度为4-37℃(最适温度为20℃)、pH为5.0-9.0(最适pH为7)、盐度为0-5％(大于5％不生长)，该菌株为兼性厌氧菌，无运动特性，可水解Tween 40(表3)。菌株的DNA G+C摩尔比例为31.6％，主要呼吸醌为MK-6，主要脂肪酸为C16:1ω6c、C16:1ω7c、iso-C15:0、iso-C17:03OH、C16:0、C16:1ω5c、iso-C17:1I/anteiso-C17:1B(表4)。
表1菌株API 20NE(非肠道革兰氏阴性杆菌鉴定系统)反应
Figure PCTCN2015089320-appb-000001
Figure PCTCN2015089320-appb-000002
注：“+”表示阳性，“－”表示阴性。
表2菌株API ZYM(酶的活性)检测
酶的种类 Wautersiella enshiensis
对照 －
碱性磷酸盐酶 +
酯酶(C4) +
类脂酯酶(C8) +
类脂酶(C14) －
白(亮)氨酸芳胺酶 +
缬氨酸芳胺酶 w
胱氨酸芳胺酶 －
胰蛋白酶 +
胰凝乳蛋白酶 －
酸性磷酸酶 +
萘酚-AS-BI-磷酸水解酶 +
α-半乳糖苷酶 －
β-半乳糖苷酶 －
β-糖醛酸苷酶 －
α-葡萄糖苷酶 +
β-葡萄糖苷酶 +
N-乙酰-葡萄糖胺酶 －
α-甘露糖苷酶 －
β-甘露糖苷酶 －
注：“+”表示阳性，“-”表示阴性，“w”表示弱阳性。
表3菌株生理生化指标
指标 Wautersiella enshiensis
温度 4～37℃
pH 5～9
盐浓度 0～5％
运动型 不运动
氧化酶 阳性
过氧化氢酶 阳性
酪蛋白水解 阴性
Twenn20水解 阴性
Twenn40水解 阳性
Twenn60水解 阴性
Twenn80水解 阴性
醌 MK-6
表4菌株脂肪酸组成特征
脂肪酸 比例(％)
C14:0 3.99
iso-C15:0 16.27
C15:1ω6c tr
C14:02OH 1.92
C16:1ω5c 7.97
C16:0 13.09
C15:0iso3OH 4.13
C15:12OH tr
iso-C17:0 1.53
C16:03OH 3.74
iso-C17:03OH 13.59
Summed Feature2 1.93
Summed Feature3 20.53
Summed Feature4 6.09
Summed Feature9 1.93
注：1：“tr”表示“含量<1％”；2：“Summed Feature 2”包含“C14:0-3OH、iso-C16:1-I”；3：“Summed feature 3”包含“C16:1ω7c、C16:1ω6c”；4：“Summed feature 4”包含“iso-C17:1I、anteiso-C17:1B”；5：“Summed feature 9”包含“iso-C17:1ω9c、C16:010-methyl”。
本发明还公开了嗜硒微生物Wautersiella enshiensis YLX-1在制备微生物转化有机硒中的应用。本发明所述微生物Wautersiella enshiensis YLX-1具有嗜硒微生物特征，在培养液中的可溶态无机硒(Se4+、Se6+)含量不超过10mg/L的条件下，可以将无机硒高效转化为含硒氨基酸(如硒代胱氨酸SeCys2)，进一步可利用这些有机硒作为硒缺乏人群的补硒来源。
本发明还公开了嗜硒微生物Wautersiella enshiensis YLX-1在制备微生物转化纳米硒中的应用。本发明所述微生物Wautersiella enshiensis YLX-1具有嗜硒微生物特征，在培养液中的可溶态无机硒(Se4+、Se6+)含量在100mg/L～6000mg/L的条件下，可以将无机硒高效转化为纳米硒，该纳米硒的尺寸为100～200nm，进一步可以利用这些具有生物活性的纳米硒作为硒缺乏人群的补硒来源。
本发明还公开了嗜硒微生物Wautersiella enshiensis YLX-1在活化含硒矿石中的应用。本发明所述微生物Wautersiella enshiensis YLX-1具有嗜硒微生物特征，可以对含硒矿石进行微生物活化，进而使含硒矿石中的不可生物利用硒活化为生物可利用硒，可将天然含硒矿石中生物可利用硒含量提高5～10倍，进一步可以将活化之后的含硒矿石加工为土壤硒改良剂，施用于缺硒土壤中种植富硒农作物。
本发明还公开了嗜硒微生物Wautersiella enshiensis YLX-1在制备富硒微生物肥料中的应用。本发明所述微生物Wautersiella enshiensis YLX-1具有嗜硒微生物特征，可以直接制作为微生物冻干粉，施用到土壤中后复水复活，活化土壤中的硒，提高土壤硒的生物可利用性，从而提高作物对硒的累积效率；也可以与有机肥复合发酵，制成微生物有机肥，施用于土壤中种植富硒农作物。
本发明还公开了嗜硒微生物Wautersiella enshiensis YLX-1在硒污染环境修复中的应用。本发明所述微生物Wautersiella enshiensis YLX-1具有嗜硒微生物特征，当污染水体中硒含量不超过100μg/L时，可以对硒污染环境中的硒进行微生物转化、累积，进一步可以收集这些微生物，一方面大大降低环境中的硒含量，达到修复环境的目的，另一方面可以利用转化、累积的微生物硒，作为微生物有机硒或微生物纳米硒的来源，用于缺硒人群的硒补充来源。其对硒污染水体中硒的清除效率可达70～80％。
借由上述方案，本发明至少具有以下优点：本发明所述嗜硒微生物菌种具有嗜硒微生物特性，可在微生物有机硒转化、微生物纳米硒合成、含硒矿石微生物活化、富硒微生物肥料制备以及硒污染环境修复中具有潜在的应用价值。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1为本发明所述菌株的形态特征；
图2为本发明所述菌株在显微镜下的形态特征；
图3为本发明菌株在0和10mg Se/L的培养液中的生长特征曲线图；
图4为本发明菌株在100、500、1000、3000和6000mg Se/L的培养液中的生长特征曲线图；
图5为本发明菌株在0、10、100、500、1000、3000和6000mg Se/L的培养液中24小时生长结束后的体系颜色特征；
图6为本发明所述菌株的16S rRNA基因序列的系统发育树示意图；
图7为本发明菌株在100mg Se/L的TSB培养液中生长48小时后的扫描电子显微镜图；
图8为本发明菌株在100mg Se/L的TSB培养液中生长48小时后的能谱分析(SEM-EDS)图；
图9为本发明菌株在6000mg Se/L的TSB培养液中生长48小时后的扫描电子显微镜图；
图10为本发明菌株在6000mg Se/L的TSB培养液中生长48小时后的能谱分析(SEM-EDS)图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
按照下述步骤进行菌种的筛选分离：
(1)初筛：无菌采集中国湖北恩施渔塘坝硒矿区渗滤水系统中的菌席沉积物样品约20g于灭菌处理的25mL的封口瓶中，平行采集3份，带回实验室4℃低温保存。然后在实验室无菌操作台内将3份平行样品进行混合均匀，取5g置于50mL灭菌蒸馏水的锥形瓶中，常温条件下150r/min震荡15min，静置30秒后取1mL菌液梯队稀释到10-3、10-4、10-5、10-6，然后接种0.05mL均匀涂布于分离培养基上(分离培养基组成：含硒200μg/kg的TSA，硒源为亚硒酸钠)，每一处理设置4组重复。常温下培养2-3天，挑取形态典型的单一菌落进行点接纯化2-3次得到纯培养物。
(2)复筛：将(1)中得到的纯培养物依次接入含硒(以亚硒酸钠配置)0、10、100、500、1000、3000、6000mg/L的TSB培养液中，均能正常生长，表 明该纯培养物具有嗜硒能力，为我们需要的嗜硒微生物。从而将该纯培养物命名为YLX-1，保藏于保藏培养基中。
对上述经复筛得到的嗜硒微生物进行形态特征观察、分子生物学鉴定和生理生化反应特征测试。
形态特征观察：该嗜硒微生物在28摄氏度的TSA培养基中培养24小时观察到的菌落为黄色平滑的圆形，并具有全缘凸面(附图1)；对菌株进行革兰氏染色，显示为革兰氏阴性菌。透射电子显微镜下观察到的菌株为棒状、非孢子形成，大小为0.4～0.7×0.8～2.0μm(附图2)；
分子生物学鉴定：从本发明所述菌株的纯培养物中提取基因组DNA，利用通用引物27f和1492r进行PCR扩增和测序，进一步通过CLUSTAL X软件和Mega 4.0软件以Neighbour-joining方法构建系统进化树(附图6)，进行系统发育分析。结果显示菌株属于沃氏菌属(Wautersiella)，但与其亲缘关系最近的Wautersiella falsenii NF 993T的序列相似性只有96.7％，结合其形态学特征，确定为沃氏菌新种，命名为Wautersiella enshiensis YLX-1，于2013年12月18日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉武汉大学，保藏编号：CCTCC No:M2013671。
生理生化反应特征测试：该本发明所述菌株的API 20NE(非肠道革兰氏阴性杆菌鉴定系统)反应显示，菌株能很好地同化葡萄糖、麦芽糖、苹果酸、柠檬酸钠，也能很好地水解七叶灵柠檬酸铁、明胶，还能与脲素和对硝基-D-甲基半乳糖进行反应(表1)。该菌株的API ZYM(酶的活性)检测显示，菌株的碱性磷酸盐酶、酯酶(C4)、类脂酯酶(C8)、白(亮)氨酸芳氨酶、胰蛋白酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶、α-葡萄糖苷酶、β-葡萄糖苷酶均具有较强的活性，缬氨酸芳胺酶具有弱活性(表2)。该菌株的生长温度为4-37℃(最适温度为20℃)、pH为5.0-9.0(最适pH为7)、盐度为0-5％(大于5％不生长)，该菌株为兼性厌氧菌，无运动特性，可水解Tween 40(表3)。菌株的DNA G+C摩尔比例为31.6％，主要呼吸醌为MK-6，主要脂肪酸为C16:1ω6c、C16:1ω7c、iso-C15:0、iso-C17:03OH、C16:0、C16:1ω5c、iso-C17:1I/anteiso-C17:1B(表4)。
实施例1
本发明所述微生物在合成有机硒中的应用。
将本发明所述微生物YLX-1菌株接种到含硒(亚硒酸钠配置)1.5mg/L的TSB培养液中，常温下200r/min培养48小时，离心收集菌体和上清液。然后利用液相色谱-原子荧光光谱联用仪(LC-GA-AFS)分别检测上清液和菌体中的硒含量与形态，结果显示上清液中几乎均为Se4+，而菌体中绝大部分为硒代胱氨酸(SeCys2)(表5)。这表明YLX-1菌株能在1.5mg Se/L条件下，将培养液中的无机硒(Se4+)转化为有机硒(SeCys2)，并累积到菌体内。进一步收集菌体可作为动物饲料等的有机硒添加，也可以做进一步的硒代氨基酸纯化原料，作为缺硒人群的有机硒补硒来源。
表5反应体系中上清液和菌体的硒形态特征
Figure PCTCN2015089320-appb-000003
注：“-”表示没有检出；“*”表示没有样品。
实施例2
本发明所述微生物在合成有机硒中的应用。
将本发明所述微生物YLX-1菌株接种到含硒(亚硒酸钠配置)10mg/L的TSB培养液中，常温下200r/min培养72小时，离心收集菌体和上清液。然后利用液相色谱-原子荧光光谱联用仪(LC-GA-AFS)分别检测上清液和菌体中的硒 含量与形态，结果显示上清液中几乎均为Se4+，而菌体中绝大部分为硒代胱氨酸(SeCys2)，在菌株的生长平稳期有少部分甲基硒代半胱氨酸(SeMeCys)和Se4+(表6)。这表明YLX-1菌株能在10mg Se/L条件下，将培养液中的无机硒(Se4+)转化为有机硒(SeCys2、SeMeCys)，并累积到菌体内。进一步收集菌体可作为动物饲料等的有机硒添加，也可以做进一步的硒代氨基酸纯化原料，作为缺硒人群的有机硒补硒来源。
表6反应体系中上清液和菌体的硒形态特征
Figure PCTCN2015089320-appb-000004
注：“-”表示没有检出；“*”表示没有样品。
实施例3
本发明所述微生物在合成纳米硒中的应用。
将本发明所述微生物YLX-1菌株接种到含硒(亚硒酸钠配置)100mg/L的TSB培养液中，常温下200r/min培养24小时，溶液出现明显的红色、浑浊。培养48小时，离心收集菌体和上清液，发现收集得到的沉淀为红色悬浮物。然后利用液相色谱-原子荧光光谱联用仪(LC-GA-AFS)和原子荧光光谱仪，利用差减法鉴定红色悬浮物的成分几乎为单质硒。进一步利用扫描电子显微镜结合能谱散射分析(SEM-EDS)，结果显示红色悬浮物中有大量菌体，在菌体表面聚集着大量尺度在100-200nm的圆球物体，EDS鉴定结果显示其为单质硒(附图7和图8)。因此收集得到的红色沉淀物应为微生物合成纳米硒。将该微生物 合成纳米硒添加进螃蟹的饲料中，结果饲喂30天后，在螃蟹的肌肉、内脏中均检出比对照组要高得多的硒含量，表明该微生物合成纳米硒可以被动物体吸收利用，是生物可利用硒，因此进一步可作为动物饲料的硒添加来源，提高动物体中的硒水平；也可以作为缺硒人群的补硒来源。
实施例4
本发明所述微生物在合成纳米硒中的应用。
将本发明所述微生物YLX-1菌株接种到含硒(亚硒酸钠配置)6000mg/L的TSB培养液中，常温下200r/min培养12小时，溶液出现明显的红色、浑浊。培养48小时，离心收集菌体和上清液，发现收集得到的沉淀为红色悬浮物。然后利用液相色谱-原子荧光光谱联用仪(LC-GA-AFS)和原子荧光光谱仪，利用差减法鉴定红色悬浮物的成分几乎为单质硒。进一步利用扫描电子显微镜结合能谱散射分析(SEM-EDS)，结果显示红色悬浮物中有大量菌体，在菌体表面聚集着大量尺度在100-200nm的圆球物体，EDS鉴定结果显示其为单质硒(附图9和图10)。因此收集得到的红色沉淀物应为微生物合成纳米硒。将该微生物合成纳米硒添加进螃蟹的饲料中，结果饲喂30天后，在螃蟹的肌肉、内脏中均检出比对照组要高得多的硒含量，表明该微生物合成纳米硒可以被动物体吸收利用，是生物可利用硒，因此进一步可作为动物饲料的硒添加来源，提高动物体中的硒水平；也可以作为缺硒人群的补硒来源。
实施例5
本发明所述微生物在活化硒矿石中的应用。
将采自湖北恩施硒矿区的硒矿石粉碎到100目，然后测定其总硒含量约为3000mg/kg，进一步通过连续提取方法，测定硒矿石中的水溶态硒约占比1％，可交换态硒约占比3％，铁锰氧化态硒约占比5％，有机结合态硒约占比10％，残留态硒占比约81％。以此硒矿粉为硒源，添加100g该硒矿粉到300mL的TSB培养液中，一组不接种YLX-1(8个平行样)，另一组接种YLX-1(8个平行样)。在室温条件下200r/min培养90天，并按照一定的时间间隔取样检测上清 液中的总硒含量(表7)，结果显示接种YLX-1菌株组中明显较对照组有更多可溶态硒进入溶液中，其硒含量是对照组的5-10倍。这表明YLX-1菌株可以将硒矿石中不可生物利用硒(如：交换态硒、铁锰氧化态硒)转化为生物可利用硒，且效率较高。进一步可利用YLX-1的这个特性对天然硒矿石进行活化，产物可作为土壤硒改良剂，用于缺硒土壤的改良。
表7反应体系上清液中硒含量特征
Figure PCTCN2015089320-appb-000005
实施例6
本发明所述微生物在富硒微生物肥料制备中的应用。
将本发明所述微生物YLX-1菌株在TSB培养液中进行50L规模化发酵培养3-5天，进一步采用冻干技术制备微生物冻干粉。该冻干粉可以直接施用到生菜作物根部土壤中，随后浇水可使冻干微生物复水复活。正常栽培30天后收获，检测显示其生菜叶中的硒含量是未施用该微生物冻干粉的生菜叶样品的3-8倍。
实施例7
本发明所述微生物在富硒微生物肥料制备中的应用。
将本发明所述微生物YLX-1菌株接种到有机肥原料中，随后进行正常的有机肥堆肥发酵，获得微生物有机肥。该微生物有机肥施用到生菜根部，正常培养30天后收获，检测显示其生菜叶中的硒含量是未施用该微生物冻干粉的生菜 叶样品的3-5倍。
实施例8
本发明所述微生物在硒污染环境修复中的应用。
测定湖北恩施硒矿区渗滤水中总硒含量约为85μg/L，并将其作为硒污染环境水体的代表，将YLX-1菌种接入300mL该水体中(8个平行样)，并加入葡萄糖作为碳源，同时设置对照组，在室温条件下200r/min培养10天，并按照一定的时间间隔取样检测上清液中的总硒含量(表8)，结果显示未接种YLX-1菌株得对照组中溶液中的硒含量基本不变，而接种YLX-1菌株处理组在菌株生长过程中明显将溶液中的硒吸收、利用了，大大降低了溶液中的硒含量，清除率达到70-80％。因此，进一步可利用YLX-1的这个特性对天然硒污染地区(如：恩施)或人工硒污染环境(如：锰冶炼区域)中的水体进行修复处理。
表8反应体系上清液中硒含量特征
Figure PCTCN2015089320-appb-000006
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种嗜硒微生物Wautersiella enshiensis YLX-1，其特征在于：该嗜硒微生物的保藏名称为沃氏菌(Wautersiella enshiensis YLX-1)，保藏单位为中国典型培养物保藏中心，保藏日期为2013年12月18日，保藏编号为CCTCCNO:M2013671。
  2. 根据权利要求1所述的嗜硒微生物Wautersiella enshiensis YLX-1，其特征在于：所述嗜硒微生物的菌株为棒状、非孢子形成，大小为0.4～0.7×0.8～2.0μm，运动方式为不运动，菌落为黄色平滑的圆形，并具有全缘凸面，革兰氏染色阴性。
  3. 根据权利要求1所述的嗜硒微生物Wautersiella enshiensis YLX-1，其特征在于：当可溶态硒含量低于10mg/L时，所述嗜硒微生物能够将该可溶态无机硒吸收转化为有机硒；当可溶态硒含量为100～6000mg/L时，所述嗜硒微生物能够将该可溶态无机硒转化为具有生物活性的纳米硒，且该纳米硒尺寸为100～200nm。
  4. 根据权利要求1至3中任一权利要求所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在制备微生物转化有机硒的应用。
  5. 根据权利要求4所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在可溶态硒含量低于10mg/L时，能够将该可溶态无机硒吸收转化为有机硒。
  6. 根据权利要求1至3中任一权利要求所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在制备微生物转化纳米硒中的应用。
  7. 根据权利要求6所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在可溶态硒含量为100～6000mg/L时，能够将该可溶态无机硒转化为具有生物活性的纳米硒。
  8. 根据权利要求1至3中任一权利要求所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在活化含硒矿石中的应用。
  9. 根据权利要求1至3中任一权利要求所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在制备富硒微生物肥料中的应用。
  10. 根据权利要求1至3中任一权利要求所述的嗜硒微生物Wautersiella enshiensis YLX-1的应用，其特征在于：所述嗜硒微生物在硒污染环境修复中的应用。
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