CN103695333B - A kind of Stenotrophomonas FQ1 bacterial strain and application - Google Patents
A kind of Stenotrophomonas FQ1 bacterial strain and application Download PDFInfo
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Abstract
The invention belongs to microbial technology field, in particular to a kind of Stenotrophomonas FQ1 bacterial strain and screening method thereof and application, the preserving number of described Stenotrophomonas FQ1 bacterial strain is CGMCC No.8272, the application method of this bacterial strain enzymolysis Enteromorpha is, by frozen FQ1 bacterial strain activation culture in LB slant medium, then the full ring of picking one is in LB liquid nutrient medium, gets 1-2mL bacterium liquid in sterilization fibre element liquid fermentation medium, shaking culture 5-7 days after shaking culture 24-28h; Again by fermented liquid low-temperature centrifugation, getting supernatant liquor is crude enzyme liquid or by filtering fermentation liquor, getting filtrate is crude enzyme liquid.Crude enzyme liquid and clean waterside lichenin are reacted under 45-55 DEG C of condition.Described Stenotrophomonas FQ1 bacterial strain has the ability of cellulase-producing, effectively the cellulose substances in Enteromorpha can be degraded to reducing sugar, and production cost is low, and equipment is simple, reaction temperature and, be applicable to large-scale commercial production.
Description
Technical field
The invention belongs to microbial technology field, particularly a kind of Stenotrophomonas FQ1 bacterial strain and screening method thereof and application.
Background technology
At present, world petroleum resource supply is gradually nervous, environmental pollution is day by day serious, and energy problem also more and more receives the concern of people.Over the past two years, the competing new forms of energy first seeking petroleum replacing of Ge great energy expenditure state, bio-ethanol fuel, with its outstanding feature of environmental protection and recyclability, was subject to attention and the active support of many countries.Bio-ethanol, as renewable resources, can reduce the consumption to oil, can directly as liquid fuel or used in combination with gasoline.
2007 start, China's Yellow Sea breaks out extensive Enteromorpha in continuous 7 years, Enteromorpha carbohydrate content analytical results shows, and its content of cellulose is 13.3%, hemicellulose level is 28.01%, starch content is 2.475%, water-soluble polysaccharide content is 11.503%, Water soluble pentosan content 11.25%.Mierocrystalline cellulose is the wire macromolecular substance be formed by connecting with β-Isosorbide-5-Nitrae-glycosidic link by glucose, is polysaccharide material the abundantest on the earth.1904, people's Late Cambrian cellulase in snail Digestive system, from then on, the research for cellulase was day by day deep.Cellulase is not single enzyme, is the general name of an enzyme system.Mierocrystalline cellulose in Enteromorpha is the main component for producing bio-ethanol, when to cellulose degradation in Enteromorpha, generally adopts commercial fibre element enzyme, but, commercial fibre element enzyme cost is higher, and cellulase solution preocess, to environment, is not suitable for large-scale commercial production.
Summary of the invention
The object of this invention is to provide a kind of Stenotrophomonas FQ1 bacterial strain, this bacterial strain can eccrine fiber element enzyme, have degradation effect to the cellulose substances in Enteromorpha, Mierocrystalline cellulose effectively can be degraded to reducing sugar, for the further recovery energy of Enteromorpha is laid a good foundation.
Another object of the present invention is to provide a kind of screening method of Stenotrophomonas FQ1 bacterial strain.
Another object of the present invention is to provide a kind of application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha.
In order to realize above technique effect, the present invention realizes by following technical solution:
A kind of Stenotrophomonas FQ1 bacterial strain, is characterized in that: preserving number is CGMCC No.8272.
The screening method of above-mentioned Stenotrophomonas FQ1 bacterial strain, its step comprises:
(1) join in Selective agar medium by the suspension of rotten Enteromorpha frond, 2-3 days cultivated by shaking table;
(2) by the bacteria suspension of selection nutrient solution after gradient dilution in step (1), be coated on differential medium, treat that bacterium colony grows;
(3) on the flat board growing bacterium colony, there is the bacterial strain of transparent circle with the screening of congo red staining method, and be separated by plate streaking partition method, purify.
In described step (1), the suspension of Enteromorpha frond rotted and the volume ratio of Selective agar medium are 1:25-45; The temperature that shaking table is cultivated is 25-35 DEG C, and rotating speed is 135-145r/min.
In described step (1), component in Selective agar medium comprises cellulose powder, SODIUMNITRATE, Repone K, Sodium phosphate dibasic, potassium primary phosphate, magnesium sulfate, yeast extract paste, hydrolyzed casein and Chen Haishui, and the proportioning that adds of each component is followed successively by 3-5g:1g:0.3-0.5g:1.1-1.3g:0.8-1g:0.4-0.5g:0.4-0.5g:0.4-0 .5g:950-1050mL.
In described step (2), component in differential medium comprises Xylo-Mucine, yeast extract paste, potassium primary phosphate, agar, murphy juice and Chen Haishui, and the proportioning that adds of each component is followed successively by 5-10g:1g:0.1-0.3g:12-16g:90-110mL:950-1050mL.
The application method of above-mentioned Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha:
A (), by Stenotrophomonas FQ1 bacterial strain shaking culture 22-26 hour in LB liquid nutrient medium, then adds frozen damping fluid in bacterium liquid, in-20 DEG C--25 DEG C preservations;
B Stenotrophomonas FQ1 bacterial strain in step (a) slant activation in LB slant medium is cultivated by (), then the full ring of picking one is in LB liquid nutrient medium, 1-2mL bacterium liquid is got in the element of sterilization fibre liquid fermentation medium, shaking culture 5-7 days after shaking culture 24-28h; By fermented liquid low-temperature centrifugation, getting supernatant liquor is crude enzyme liquid or by filtering fermentation liquor, getting filtrate is crude enzyme liquid;
C () is dry by clean Enteromorpha, pulverizing, then under 45-55 DEG C of condition, reacts 45-50 hour with the crude enzyme liquid in step (b).
In described step (a), the temperature of shaking culture is 25-35 DEG C, and rotating speed is 135-145r/min; The glycerine of described damping fluid to be volumetric concentration be 45-55%, the volume ratio of damping fluid and LB substratum is 1:1-1.5.
In described step (b), the temperature of shaking culture is 30-35 DEG C, and rotating speed is 135-145r/min; Centrifugation temperature is 3-5 DEG C, and centrifugal rotational speed is 3500-4500r/min, and centrifugation time is 10-20 minute.
In described step (b), the component in cellulosic pulp fermention medium comprises yeast extract, peptone, Xylo-Mucine and Chen Haishui, and the proportioning that adds of each component is followed successively by 1g:3-6g:8-12g:950-1050mL.
In described step (c), by clean Enteromorpha dry 10-14 hour at 55-65 DEG C, be then crushed to 70-90 order; Enteromorpha is 1g:45-55mL with the add-on ratio of crude enzyme liquid.
The Chen Haishui used in above-mentioned steps collects from urban beach marine site, Discharge Engineering in Shanghai Jinshan District.
The present invention screens the bacterial strain obtained and belongs to Stenotrophomonas genus (Stenotrophomonas sp.), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.8272, its 16S rDNA sequence, as follows:
CTGGGTGGCGAGTGGCGGACGGGTGAGGAATACATCGGAATCTACTTTTTCGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGCAGGGGATCTTCGGACCTTGCGCGATTGAATGAGCCGATGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATACCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAATCCAGCTGGCTAATACCCGGTTGGGATGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTCGTTTAAGTCCGTTGTGAAAGCCCTGGGCTCAACCTGGGAACTGCAGTGGATACTGGGCGACTAGAGTGTGGTAGAGGGTAGCGGAATTCCTGGTGTAGCAGTGAAATGCGTAGAGATCAGGAGGAACATCCATGGCGAAGGCAGCTACCTGGACCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAGCCGGCGACGGTAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCCGATGACCGGG
The invention has the beneficial effects as follows: 1, Stenotrophomonas FQ1 bacterial strain provided by the invention can eccrine fiber element enzyme, and when applying, directly the liquid fermenting system of this bacterial strain and enteromorpha fiber element are acted on, can be reducing sugar by the cellulose degradation in Enteromorpha effectively, ready for further preparing bio-ethanol.2, the present invention is with green damp algae Enteromorpha for raw material, turn waste into wealth, and production cost is low, and equipment is simple, reaction temperature and, easy and simple to handle, be applicable to large-scale commercial production.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Chen Haishui: refer to and gather seawater for subsequent use in a large number by urban beach marine site, Discharge Engineering in Shanghai Jinshan District.
Embodiment 1
The screening of Stenotrophomonas FQ1 bacterial strain
(1) picking up from natural condition growth and the intensive floating rotten Enteromorpha frond in green tidal sea territory respectively, is locality Qingdao No.One Bathing Beach and the 6th bathing beach.Take back laboratory with insulation can low-temperature transport after gathering and carry out bacteria screening.
By the rotten Enteromorpha frond suspension 1mL collected, add in the Erlenmeyer flask that 30mL Selective agar medium is housed, be fixed on by Erlenmeyer flask on shaking table, 30 DEG C, 140r/min shakes cultivation 2 ~ 3 days, until nutrient solution becomes muddy.
Wherein Selective agar medium is: cellulose powder 5g,
SODIUMNITRATE 1g,
Repone K 0.5g,
Sodium phosphate dibasic 1.2g,
Potassium primary phosphate 0.9g,
Magnesium sulfate 0.5g,
Yeast extract paste 0.5g,
Hydrolyzed casein 0.5g,
Chen Haishui 1000mL.
(2) will the substratum after cultivating be selected to carry out gradient dilution: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, be 10 by extent of dilution
-5, 10
-6bacteria suspension respectively get 0.2mL and be coated on differential medium, observe bacterium colony after 48h.
Wherein differential medium is: Xylo-Mucine 5-10g,
Yeast extract paste 1g,
Potassium primary phosphate 0.25g,
Murphy juice 100mL,
Agar 15g,
Chen Haishui 1000mL.
(3) the differential medium flat board after growing bacterium colony drips the Congo red solution-dyed of 1%, unnecessary Congo red solution is removed after 15min, then the sodium chloride solution of 1mol/L is added, swing surface Congo red, solution is removed after 15min, observe transparent circle, measure the diameter of transparent circle, bacterial strain larger for transparent circle is carried out plate streaking separation and obtain pure bacterial strain.
Embodiment 2
Producing of crude enzyme liquid.
(1) by the Stenotrophomonas FQ1 that obtains in embodiment 1 in LB liquid nutrient medium, 28 DEG C, 140r/min cultivates 24h, then in bacterium liquid, adds frozen damping fluid.The volumetric concentration of damping fluid is the glycerine of 50%, and the volume ratio that adds of damping fluid and bacterium liquid is 1:1, adds frozen damping fluid, in order to protect Stenotrophomonas FQ1 bacterial strain better, makes this bacterial strain can not be dead because of dehydration.
(2) slant activation in LB substratum of the Stenotrophomonas FQ1 bacterial strain in above-mentioned is cultivated, after activating for 2 generations, the full ring of picking one is in LB liquid nutrient medium, 28 DEG C, 140r/min cultivates and to get after 24h in cellulosic pulp fermention medium that 1mL cools in 50mL sterilizing, 35 DEG C, 140r/min shaking culture 6 days.
Wherein liquid fermentation medium is:
Yeast extract 1g,
Peptone 5g,
Xylo-Mucine 10g,
Chen Haishui 1000ml.
(3) by the liquid fermentation and culture liquid that obtains in step (2) in 4 DEG C, centrifugal 15 minutes of 4000r/min, gets supernatant liquor as crude enzyme liquid.
The determination of activity of crude enzyme liquid:
Filter paper enzyme activity (FPA) measures: Xinhua's No. 1 filter paper is cut into 1 × 6cm size and is rolled into rouleau, put in 25mL colorimetric cylinder, add 1mL citrate buffer solution (pH=4.8), add crude enzyme liquid obtained in 1mL the present embodiment again, shake up and make filter paper be fully immersed in solution, press DNS method immediately after 50 DEG C of accurate insulation 1h and survey reducing sugar, recording enzyme activity is 20.27U/mL.
CMC enzyme activity determination: pipette the crude enzyme liquid of gained in 1mL the present embodiment in 25mL colorimetric cylinder, add the citrate buffer solution (pH=4.4) of 1mL containing 0.5%CMC-Na, press DNS method immediately after 50 DEG C of accurate insulation 30min and survey reducing sugar, recording enzyme activity is 44.32U/mL.
Embodiment 3
Producing of crude enzyme liquid.
Step (1) and step (2) are with embodiment 2.
(3) the liquid fermentation and culture liquid obtained in step (2) is filtered, get its filtering solution as crude enzyme liquid.
The Activity determination of crude enzyme liquid:
Filter paper enzyme activity (FPA) measures: measuring method is with embodiment 2, and recording enzyme activity is 24.90U/mL.
CMC enzyme activity determination: measuring method is with embodiment 2, and recording enzyme activity is 49.28U/mL.
Embodiment 4
Crude enzyme liquid is applied the enzymolysis of Enteromorpha.
(1) the green damp algae Enteromorpha broken out in a large number with Yellow Sea is for raw material, and after foreign material such as cleaning removing shell, sandstone etc., clear water rinsing marine alga 3-4 time, is positioned over 12h in 60 DEG C of baking ovens, pulverizes 1 minute after oven dry with pulverizer, for subsequent use after crossing 80 mesh sieves.
(2) the waterside lichenin of 1g drying is placed in triangular flask, join in crude enzyme liquid obtained in embodiment 3, the proportioning that adds of waterside lichenin and crude enzyme liquid is 1g:50mL, 48h is reacted in 50 DEG C of water-baths, then press DNS method, recording Reducing sugar is 9.1%, illustrates that crude enzyme liquid has Degradation well to the cellulose substances in Enteromorpha, can be reducing sugar by cellulose degradation, produce bio-ethanol further for Enteromorpha and get ready.
Embodiment 5
Crude enzyme liquid is applied the enzymolysis of Enteromorpha.
(1) the green damp algae Enteromorpha broken out in a large number with Yellow Sea is for raw material, and after foreign material such as cleaning removing shell, sandstone etc., clear water rinsing marine alga 3-4 time, is positioned over 12h in 60 DEG C of baking ovens, pulverizes 1 minute after oven dry with pulverizer, for subsequent use after crossing 80 mesh sieves.
(2) the waterside lichenin of 1g drying is placed in triangular flask, join in crude enzyme liquid obtained in embodiment 2, the proportioning that adds of waterside lichenin and crude enzyme liquid is 1g:50mL, 48h is reacted in 50 DEG C of water-baths, then press DNS method, recording Reducing sugar is 7.7%, illustrates that crude enzyme liquid has Degradation well to the cellulose substances in Enteromorpha, can be reducing sugar by cellulose degradation, produce bio-ethanol further for Enteromorpha and get ready.
Claims (6)
1. Stenotrophomonas strain (Stenotrophomonas sp.) FQ1, is characterized in that: preserving number is CGMCC No.8272.
2. the application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha as claimed in claim 1:
A (), by Stenotrophomonas FQ1 bacterial strain shaking culture 22-26 hour in LB liquid nutrient medium, then adds frozen damping fluid in bacterium liquid, in-20 DEG C--25 DEG C preservations;
B Stenotrophomonas FQ1 bacterial strain in step (a) slant activation in LB slant medium is cultivated by (), then the full ring of picking one is in LB liquid nutrient medium, 1-2mL bacterium liquid is got in the element of sterilization fibre liquid fermentation medium, shaking culture 5-7 days after shaking culture 24-28h; By fermented liquid low-temperature centrifugation, getting supernatant liquor is crude enzyme liquid or by filtering fermentation liquor, getting filtrate is crude enzyme liquid;
C () is dry by clean Enteromorpha, pulverizing, then under 45-55 DEG C of condition, reacts 45-50 hour with the crude enzyme liquid in step (b).
3. the application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha according to claim 2, it is characterized in that: in described step (a), the temperature of shaking culture is 25-35 DEG C, and rotating speed is 135-145r/min; The glycerine of described damping fluid to be volumetric concentration be 45-55%, the volume ratio of damping fluid and bacterium liquid is 1:1-1.5.
4. the application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha according to claim 2, it is characterized in that: in described step (b), the temperature of shaking culture is 30-35 DEG C, and rotating speed is 135-145r/min; Centrifugation temperature is 3-5 DEG C, and centrifugal rotational speed is 3500-4500r/min, and centrifugation time is 10-20 minute.
5. the application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha according to claim 2, it is characterized in that: in described step (b), component in cellulosic pulp fermention medium comprises yeast extract, peptone, Xylo-Mucine and Chen Haishui, and the proportioning that adds of each component is followed successively by 1g:3-6g:8-12g:950-1050mL.
6. the application method of Stenotrophomonas FQ1 bacterial strain enzymolysis Enteromorpha according to claim 2, is characterized in that: in described step (c), by clean Enteromorpha dry 10-14 hour at 55-65 DEG C, is then crushed to 60-100 order; Enteromorpha is 1g:45-55mL with the add-on ratio of crude enzyme liquid.
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| CN104877944B (en) * | 2015-06-14 | 2016-02-03 | 中国海洋大学 | The strain of a kind of astaxanthin ester enzyme-producing bacteria and the application in free astaxanthin preparation thereof |
| CN106282070B (en) * | 2016-10-08 | 2019-06-21 | 南开大学 | The screening and application of one plant of polybrominated diphenyl ethers (PBDEs) degradation bacteria |
| CN109536413B (en) * | 2018-12-21 | 2020-06-23 | 浙江工业大学 | Stenotrophomonas HY-2 and application thereof in degradation of organic matters |
| CN111471617A (en) * | 2020-04-08 | 2020-07-31 | 陕西省微生物研究所 | Cellulose degradation composite flora and screening thereof and application of saprophytic fungi residue for decomposing |
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