CN111982875A - Method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis - Google Patents

Method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis Download PDF

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CN111982875A
CN111982875A CN202010844387.4A CN202010844387A CN111982875A CN 111982875 A CN111982875 A CN 111982875A CN 202010844387 A CN202010844387 A CN 202010844387A CN 111982875 A CN111982875 A CN 111982875A
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solution
screening
spectrum analysis
dimensional fluorescence
fluorescence spectrum
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柴立伟
郑维爽
黄艺
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Peking University Shenzhen Graduate School
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

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Abstract

The invention belongs to the technical field of rapid screening of polyhydroxyalkanoate-producing bacteria, and provides a method for screening polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis. The screening method of the invention comprises the following steps: (1) taking a bacterium solution to be detected to separate a culture solution to obtain a concentrated bacterium solution, adjusting the concentration of the concentrated bacterium solution, and then carrying out freeze drying to obtain freeze-dried bacterium powder; (2) adding normal hexane into the freeze-dried bacterial powder prepared in the step (1), carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected; (3) and (3) carrying out three-dimensional fluorescence spectrum analysis on the sample to be detected in the step (2). Compared with the traditional gas chromatography analysis method, the screening method provided by the invention is labor-saving and labor-saving, greatly improves the screening efficiency of the strains, and is a brand-new screening method for producing the polyhydroxyalkanoate bacteria.

Description

Method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis
Technical Field
The invention relates to the technical field of rapid screening of polyhydroxyalkanoate producing bacteria, in particular to a method for screening polyhydroxyalkanoate producing bacteria based on three-dimensional fluorescence spectrum analysis.
Background
Polyhydroxyalkanoates (PHA) are a kind of functional polyesters with similar structures in biological cells, and widely exist in cells of microorganisms as energy storage substances. PHA is a polymer material that has mechanical and thermoplastic properties similar to those of polyethylene and polypropylene, and thus can be developed and utilized. Besides, PHA has biodegradability and biocompatibility, so that PHA is considered to be an environment-friendly bio-based polymer material, and has important application prospects in the aspects of packaging industry, organic agriculture, medical materials and advanced sensor development.
As a polymer, the PHA has different mechanical properties due to different structural monomers, monomer arrangement modes and polymerization degrees, and has different application scenes. Current industrial-grade PHA production is highly dependent on microbial species that produce PHA efficiently. It has been shown that different types of microorganisms may produce different types of PHA. Therefore, large-scale microbial strain screening is an important way for modifying PHA materials, accelerating industrial production capacity and reducing production cost in the future.
With the development of molecular biology technology, PHA-producing strains can be primarily screened by identifying the expression level of PHA synthesis genes. However, genotype is not equal to phenotype, and the PHA-producing ability of the bacteria needs to be determined quantitatively or semi-quantitatively after culture. Currently, the most used method for quantification of PHA is gas chromatography. The gas chromatography has the advantages of accurate quantification and capability of providing complex material composition information, and has the disadvantages of time and labor waste in sample extraction, long-time sample pretreatment process required for chromatographic analysis, high requirements on the proficiency of operators and unsuitability for large-scale screening. On the other hand, the lipophilic fluorescent dye nile red can be combined with PHA to emit fluorescence in an organic solvent so as to qualitatively judge whether the bacteria produce PHA. However, this method is less sensitive and, particularly in cases where current culture conditions are not conducive to bacterial accumulation of PHA, screen leakage may occur. Three-dimensional fluorescence spectrum analysis is a new organic matter detection method which is started in the field of environmental chemistry in recent years and is widely applied to the related research of natural source organic matters and microbial analytes. Different from the traditional fluorescence detection method, the three-dimensional fluorescence spectrum can provide the fluorescence characteristics of continuous emission light of a sample on continuous excitation light, has the advantages of high resolution and high resolution, and has wide application in materials science and environmental science. However, the current research shows that the self-fluorescence characteristics of PHA are not obvious and are not suitable for fluorescence analysis.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis, which comprises the following steps:
(1) taking a bacterium solution to be detected to separate a culture solution to obtain a concentrated bacterium solution, adjusting the concentration of the concentrated bacterium solution, and then carrying out freeze drying to obtain freeze-dried bacterium powder;
(2) adding normal hexane into the freeze-dried bacterial powder prepared in the step (1), carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected;
(3) and (3) carrying out three-dimensional fluorescence spectrum analysis on the sample to be detected in the step (2).
Preferably, the method for adjusting the concentration of the concentrated bacterial liquid is to add sterile water into the concentrated bacterial liquid and adjust the OD value of the concentrated bacterial liquid at 600nm to be 1.5-1.8.
Preferably, the temperature of the freeze drying is-50 to-70 ℃, and the time is 20 to 28 hours.
Preferably, the volume ratio of the n-hexane to the bacterial liquid to be detected is 1: 0.8-1.5.
Preferably, the power of ultrasonic extraction is 300-500W, and the time of ultrasonic extraction is 3-8 min.
Preferably, the addition amount of the nile red staining solution is 0.01-0.05% of the volume of the bacterial solution to be detected.
Preferably, the concentration of the nile red staining solution is 0.005-0.015 mg/mL.
Preferably, the three-dimensional fluorescence spectrum analysis conditions are as follows: the wavelength range of the detection excitation light is 200-600 nm, the step is 2nm, the wavelength of the detection emission light is 246.977-824.286 nm, the step is 4nm, and the detection integration time is 0.1 s.
The invention provides a method for screening PHA-producing bacteria based on three-dimensional fluorescence spectrum analysis, which relates to screening and detection of microbial resources of environment-friendly bio-based polymer materials (PHA), and the main principle is that the PHA potential yield of microorganisms is evaluated based on the intensity of fluorescence by utilizing the fact that Nile red can emit strong fluorescence in a specific wavelength region after being combined with PHA. Compared with the traditional meteorological chromatographic analysis, the screening method provided by the invention is labor-saving and labor-saving, greatly improves the screening efficiency of the bacteria, and is a brand-new screening method for PHA-producing bacteria.
Drawings
FIG. 1 is an electrophoretogram of the product components of the strain N.aquimarinus TN28-1 of example 1;
FIG. 2 three-dimensional fluorescence characteristic peaks of pure PHB in example 1 and P.putida KCTC1751TAnd a three-dimensional fluorescence characteristic peak of the product component of the N.aquimarinus TN28-1 strain;
FIG. 3 shows the time of peak of pure PHB and P.pudida KCTC1751 in comparative example 1TAnd the peak emergence time of the product component of the N.aquimarinus TN28-1 strain;
FIG. 4 shows the pure PHB and P.putida KCTC1751 of comparative example 1TAnd similarity of gas chromatography peak area and three-dimensional fluorescence integral volume of PHB produced by the strain N.aquimarinus TN 28-1.
FIG. 5 is P.putida KCTC1751 in comparative example 2TAnd the cell morphology of the N.aquimarinus TN28-1 bacterium under a fluorescence microscope (pure PHB cell-free morphology, so no comparison).
Detailed Description
The invention provides a method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis, which comprises the following steps:
(1) taking a bacterium solution to be detected to separate a culture solution to obtain a concentrated bacterium solution, adjusting the concentration of the concentrated bacterium solution, and then carrying out freeze drying to obtain freeze-dried bacterium powder;
(2) adding normal hexane into the freeze-dried bacterial powder prepared in the step (1), carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected;
(3) and (3) carrying out three-dimensional fluorescence spectrum analysis on the sample to be detected in the step (2).
The method comprises the steps of separating a culture solution from a bacterial solution to be detected to obtain a concentrated bacterial solution, adjusting the concentration of the concentrated bacterial solution, and then carrying out freeze drying to obtain freeze-dried bacterial powder.
In the invention, the method for separating the culture solution is preferably centrifugation, and the centrifugation condition is preferably 10000-15000 Xg, more preferably 11000-14000 Xg, and even more preferably 13000 Xg; the time for the centrifugation is preferably 1 min.
In the present invention, the method of adjusting the concentration of the concentrated bacterial liquid is preferably: sterile water is added into the concentrated bacterial liquid, and the OD value of the concentration at 600nm is preferably 1.5-1.8, more preferably 1.6-1.7, and still more preferably 1.6.
In the present invention, the concentrated bacterial liquid after concentration adjustment is preferably placed in a sealable glass test tube and then freeze-dried.
In the present invention, the temperature of the freeze-drying is preferably-50 to-70 ℃, more preferably-55 to-65 ℃, and still more preferably-60 ℃; the time is preferably 20 to 28 hours, more preferably 22 to 25 hours, and still more preferably 24 hours.
And after the freeze-dried bacterial powder is obtained, adding normal hexane into the freeze-dried bacterial powder, carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected.
In the invention, the volume ratio of the n-hexane to the bacterial liquid to be detected is preferably 1: 0.8-1.5, more preferably 1: 0.9-1.2, and still more preferably 1: 1.
In the invention, the power of ultrasonic extraction is preferably 300-500W, more preferably 360-440W, and still more preferably 400W; the ultrasonic extraction time is preferably 3-8 min, more preferably 4-7 min, and still more preferably 5 min.
In the invention, the addition amount of the nile red staining solution is preferably 0.01 to 0.05% of the volume of the bacterial solution to be detected, more preferably 0.02 to 0.03%, and even more preferably 0.02%.
In the invention, the nile red staining solution is prepared by dissolving nile red in acetone.
In the invention, the concentration of the nile red staining solution is preferably 50 to 150ng/mL, more preferably 70 to 120ng/mL, and even more preferably 100 ng/mL.
And after a sample to be detected is prepared, three-dimensional fluorescence spectrum analysis can be carried out.
In the present invention, the three-dimensional fluorescence spectrum analysis conditions are as follows: the wavelength range of the detection excitation light is preferably 200-600 nm, the step is preferably 2nm, the wavelength of the detection emission light is preferably 246.977-824.286 nm, the step is preferably 4nm, and the detection integration time is preferably 0.1 s.
And after the fluorescence intensity of the sample is measured, comparing the fluorescence quantity of the sample with the background fluorescence quantity, and preliminarily evaluating the PHA production capacity of the strain.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
PHB (poly-3-hydroxyvalerate) is the polymer with the simplest structure, the most studied and the most thorough structure in all PHA substances, and the structural unit of the polymer is trihydroxy valerate. This example characterizes the ability of the method to detect substances with similar physicochemical properties, PHA, by directly detecting PHB.
This example separately deals with pure PHB and a commercial PHA-producing bacterium Pseudomonas putida KCTC1751TAnd performing three-dimensional fluorescence spectrum analysis on the product components of the PHA-producing strain Nitratireducor aquimarinus TN28-1 subjected to environmental screening.
In this example, pure PHB was purchased commercially; strain p.putida KCTC1751TPurchased from Korean Collection for Type Cultures, No. KCTC1751, which is now proven to be a PHA-producing microorganism (PHB is the main component of PHA) with high efficiency, widely used in industrial production; the strain N.aquimarinus TN28-1 is screened from the sediment of Shenzhen Dapeng Bay sea area, and the Collection number is MCCC 1K04607(MCCC, Marine Culture Collection of China Marine microorganism Culture Collection management center). Amplification Using Polymerase Chain Reaction (PCR)phaC synthetase gene, the primer is: phaC1F1(5 '-TGGARCTGATCCAGTAC-3') and phaC1R1(5 '-CGGGTTGAGRATGCTCTG-3'), with the provisos that: 5min at 94 ℃, then 1min at 94 ℃, 1min at 54 ℃, 1min at 72 ℃ for 30 cycles, and finally 10min at 72 ℃. The PCR products were separated by agarose gel electrophoresis, and separated for 15min at 120V using 1% agarose, and the gel pattern (as shown in FIG. 1) confirmed that there was a clear band at 500bp, demonstrating the potential of PHA production of this strain.
In this example, the preparation method of the pure PHB sample to be tested is as follows: firstly, 3mL of n-hexane solution is added into a quartz fluorescent cuvette with a plug, 1 mu L of pure PHB is absorbed by a micropipette and added into the cuvette, and the cuvette is covered and evenly mixed. Then, 1. mu.L of Nile Red staining solution (100 ng/mL in acetone) was added, followed by detection using a three-dimensional fluorescence spectroscopy method.
Meanwhile, the nutrient agar culture medium is used for respectively treating P.putida KCTC1751TAnd N.aquimarinus TN 28-1. Wherein the nutrient agar culture medium comprises the following components in concentration: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.3. Respectively culturing the culture medium to obtain P.putida KCTC1751 to be detectedTBacterial liquid and N.aquimarinus TN28-1 bacterial liquid to be detected.
Taking P.putida KCTC1751 to be detectedTCentrifuging the bacterial solution at 13000 Xg for 1min, and separating the culture medium to obtain concentrated bacterial solution; then, the concentration of the concentrated bacterial liquid was adjusted with sterile water so that the OD at 600nm was 1.6. 5mL of concentrated bacterial liquid with the adjusted concentration is filled into a sealable glass test tube, and is frozen and dried for 24h at the temperature of minus 60 ℃ to obtain freeze-dried bacterial powder; adding 5mL of n-hexane into a sealable glass test tube containing the lyophilized powder, sealing the container, and ultrasonically extracting at 400W for 5 min. Then adding 1 mul of nile red dye solution with the concentration of 100ng/mL to obtain P.putida KCTC1751TA sample to be tested for bacteria. Similarly, a sample to be tested of the N.aquimarinus TN28-1 strain is prepared according to the method, and three-dimensional fluorescence spectrum analysis is carried out.
Pure PHB, P.putida KCTC1751 were subjected to the following parametersTA sample to be tested of the bacteria and a sample to be tested of the N.aquimarinus TN28-1 bacteriaAnd (6) analyzing the rows. The detection of the relevant parameters is as follows: the sample detection amount is 3 mL. The wavelength range of the detection excitation light is 200-600 nm, the step is 2nm, the wavelength of the detection emission light is 246.977-824.286 nm, the step is 4nm, and the detection integration time is 0.1 s. In the detection, 1 μ L of nile red staining solution (with a mass concentration of 100ng/mL and acetone as a solvent) is added into a blank n-hexane solvent (with a chromatographic purity of 95%) as a fluorescent blank control. The amount of fluorescence generated by the blank as a reaction was subtracted after the sample detection, and the amount of fluorescence was used to evaluate the PHA yield of the strain. The resulting data were visualized and quantitatively analyzed using drEEM tool. In this example, the three-dimensional fluorescence spectrum analysis used an Aqualog simultaneous absorption-three-dimensional fluorescence detection scanning spectrometer from HORIBA.
The results are shown in FIG. 2. Commercially pure PHB exhibits a significant characteristic peak in fluorescence at Ex 500nm and Em 550nm, with a peak height of about 700 a.u.; pudida KCTC1751TThe characteristic peak of the fluorescence of the bacteria is the same as that of pure PHB, the positions of the characteristic peak are 500nm for Ex, 550nm for Em, and the peak height is 450 A.U.; the fluorescence characteristic peak position of the N.aquimarinus TN28-1 bacterium is similar to that of PHB, and is mainly located at 490nm, 530nm and 120A.U. of peak height. Comparing the peak heights of the two strains at the position of the characteristic peak by using the three-dimensional fluorescence spectrum analysis result, PTHas higher PHB yield, and compared with the yield of N.aquimarinus TN28-1, the yield is lower.
Comparative example 1
To further verify the accuracy of the three-dimensional fluorescence spectroscopy analysis, commercially purchased pure PHB was analyzed using a gas chromatograph-mass spectrometer with a PHB peak time of 3.65min in the gas chromatography analysis (as shown in fig. 3). And performing gas chromatography-mass spectrometry on the components generated by the two strains to be detected. The detection method comprises the following steps: respectively taking 5mL of fermentation liquor of P.putida KCTC1751 bacteria and N.aquimarinus TN28-1 bacteria, centrifuging for 20min at the speed of 5000 Xg, removing supernate, respectively adding 1mL of chloroform and 1mL of methanol solution containing 15% (v/v) concentrated sulfuric acid, fully mixing uniformly, carrying out oil bath at 100 ℃ for 150min, and carrying out methyl esterification reaction under an acidic condition. After the reaction is finished, the mixture is cooled for 5min in an ice bath, 1mL of deionized water is respectively added, the mixture is fully and uniformly mixed for 30s, the mixture is kept stand for 1min, and 150 mu L of lower-layer organic phase is taken out and placed into a GC small tube for chromatographic analysis. As shown in FIG. 3, the peak appearance time of the product of P.putida KCTC1751 strain was 3.587min and the peak appearance time of the product of N.aquimarinus TN28-1 strain was 3.650min in gas chromatography-mass spectrometry. Meanwhile, PHB characteristic peaks of the gas chromatography and the three-dimensional fluorescence spectrum analysis are integrated, and the integration result is shown in fig. 4, wherein fig. 4A and 4B respectively compare the gas chromatography peak area and the three-dimensional fluorescence integration volume of three samples, and fig. 4C represents the consistency of the results of the two methods in the form of a scatter diagram. As can be seen from FIG. 4, the PHB characteristic peak integration result of the three-dimensional fluorescence has stronger consistency with the traditional gas chromatography analysis result. Therefore, three-dimensional fluorescence spectroscopy can be demonstrated as a potential PHA yield detection method. In addition, the results of example 1 and comparative example 1 show that it is feasible to detect the fluorescence characteristic peak of the lipophilic dye after binding with PHB in an organic solvent environment using three-dimensional fluorescence spectroscopy.
Comparative example 2
The accuracy of the three-dimensional fluorescence spectrum analysis is further verified by a PHA fluorescence reaction observation method. The PHA fluorescence reaction observation steps are as follows: respectively taking 1mL of strain fermentation liquor of P.putida KCTC1751 strain and N.aquimarinus TN28-1 strain, centrifuging at 13000 Xg for 1min, and removing supernatant. To the remaining cells, 1. mu.L of Nile Red staining solution (10. mu.g/mL DMSO (dimethyl sulfoxide)) was added and mixed well, and 1.5. mu.L of Nile Red staining solution was added dropwise and suspended on a cleaned slide glass, dried for 5 seconds, and carefully covered with a cover glass on the surface. Cell morphology was photographed using a fluorescence microscope at brightfield and excitation 562nm, respectively, emitting 594nm fluorescence.
Similar results to the three-dimensional fluorescence analysis can also be obtained by comparing the cell morphology of the two species under a fluorescence microscope (as shown in FIG. 5). And after nile red staining, the PHA production capacity of N.aquimarinus TN28-1 is difficult to determine under a fluorescence microscope, which shows that the three-dimensional fluorescence spectrum analysis method is superior to common staining and has higher precision.
In conclusion, the invention provides the PHA-producing strain screening method based on three-dimensional fluorescence spectrum analysis, which can avoid the time-consuming and labor-consuming processes of the traditional meteorological chromatographic analysis, greatly improve the strain screening efficiency, and has higher accuracy compared with a fluorescence reaction observation method.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (8)

1. A method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis is characterized by comprising the following steps:
(1) taking a bacterium solution to be detected to separate a culture solution to obtain a concentrated bacterium solution, adjusting the concentration of the concentrated bacterium solution, and then carrying out freeze drying to obtain freeze-dried bacterium powder;
(2) adding normal hexane into the freeze-dried bacterial powder prepared in the step (1), carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected;
(3) and (3) carrying out three-dimensional fluorescence spectrum analysis on the sample to be detected in the step (2).
2. The screening method according to claim 1, wherein the method for adjusting the concentration of the concentrated bacterial liquid comprises: and adding sterile water into the concentrated bacterial liquid, and adjusting the OD value of the concentrated bacterial liquid at 600nm to be 1.5-1.8.
3. The screening method according to claim 1 or 2, wherein the freeze-drying temperature is-50 to-70 ℃ and the time is 20 to 28 hours.
4. The screening method according to claim 3, wherein the volume ratio of the n-hexane to the bacterial liquid to be tested is 1: 0.8-1.5.
5. The screening method according to claim 1, wherein the power of the ultrasonic extraction is 300-500W, and the time of the ultrasonic extraction is 3-8 min.
6. The screening method according to claim 1, wherein the nile red staining solution is added in an amount of 0.01 to 0.05% by volume of the bacterial solution to be tested.
7. The screening method according to claim 1 or 6, wherein the concentration of the Nile Red staining solution is 50 to 150 ng/mL.
8. The screening method according to claim 1, wherein the conditions of the three-dimensional fluorescence spectrum analysis are: the wavelength range of the detection excitation light is 200-600 nm, the step is 2nm, the wavelength of the detection emission light is 246.977-824.286 nm, the step is 4nm, and the detection integration time is 0.1 s.
CN202010844387.4A 2020-08-20 2020-08-20 Method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis Pending CN111982875A (en)

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CN104520433A (en) * 2012-04-11 2015-04-15 赫姆霍尔兹传染病研究中心有限责任公司 PHA-producing genetically engineered microorganisms
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