WO2018041215A1 - Procédé et dispositif de détection rapide d'organismes de 10 à 50 μm dans de l'eau de ballast de navire - Google Patents

Procédé et dispositif de détection rapide d'organismes de 10 à 50 μm dans de l'eau de ballast de navire Download PDF

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WO2018041215A1
WO2018041215A1 PCT/CN2017/099893 CN2017099893W WO2018041215A1 WO 2018041215 A1 WO2018041215 A1 WO 2018041215A1 CN 2017099893 W CN2017099893 W CN 2017099893W WO 2018041215 A1 WO2018041215 A1 WO 2018041215A1
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filter
sample
liquid
ballast water
filtrate
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PCT/CN2017/099893
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English (en)
Chinese (zh)
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张灿
王建
詹世杰
董晓林
靳双亭
韩德民
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威海中远造船科技有限公司
张灿
王建
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Publication of WO2018041215A1 publication Critical patent/WO2018041215A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator 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
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the invention relates to a method and a device for detecting a biological living body in a ballast water carried by a ship, and in particular relates to a method and a device for detecting a 10-50 um biological rapid detection in a ship ballast water with high detection precision and reliable data.
  • Surviving organisms having a minimum size greater than or equal to 50 ⁇ m are less than 10/m3; less than 10 ⁇ g/ml of living organisms having a minimum size of less than 50 ⁇ m but greater than or equal to 10 ⁇ m;
  • the indicator microorganism is less than the following concentrations:
  • Toxic Vibrio cholerae (serotype 01 and 0139) is less than 1 cfu / 100 ml;
  • E. coli less than 250 cfu / 100 ml
  • Enterococci less than 100 cfu / 100ml.
  • the object of the present invention is to solve the above-mentioned deficiencies of the prior art, and provide a 10-50um biological rapid detection method and device for ship ballast water with simple structure, convenient use, high detection speed, high precision and reliable data.
  • a 10-50um biological rapid detection method for ship ballast water characterized in that the method comprises the following steps:
  • the 10 um surviving organism is filtered out with the liquid;
  • the method for obtaining the relative fluorescence strong reference value is as follows:
  • the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer
  • each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained
  • the data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
  • the relative fluorescence intensity value obtained in the step (6) is compared with the relative fluorescence intensity reference value obtained in the step g, and it is judged whether the ballast water sample meets the emission standard.
  • the reference test sample described in the step a of the present invention is obtained by using a large-aperture filter to viable a living biofilter of not less than 50 ⁇ m in the ballast water sample (to be discharged) treated by the ballast water treatment apparatus. Out, the filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organism of not less than 10um in the filtrate, and the living organisms less than 10um are filtered with the liquid; using 3-10% Aml for cleaning Liquid backwashing small pore size filter, rinsing the living organism with a size of 10-50 um into the sample tank; detecting the liquid in the sample tank by a FDA-CMFDA method under a fluorescence microscope, wherein 10-50 um of living organisms When the content meets the requirements, it can be used as a reference test sample.
  • the reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 ⁇ m in the filtrate, and a living organism of less than 10 ⁇ m with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Microscopically, when the content of 10-50um surviving organisms meets the requirements, It can be used as a reference test sample.
  • UV ultraviolet
  • NaClO sodium hypochlorite
  • the large-aperture filter described in the present invention is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; the small-diameter filter is a microporous filter having an absolute pore diameter of 10 ⁇ m. a filter that can be backwashed;
  • the living cell fluorescent staining agent in the present invention is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank.
  • the staining reaction time is 5-30 (10) minutes.
  • the FDA is fluorescein diacetate.
  • the cell lysate described in the present invention is one of a mixture of methanol and chloroform (volume ratio of 1:1 to 1:3) or acetone.
  • the utility model relates to a 10-50um biological rapid detecting device for ship ballast water, which comprises a sample box, a flushing liquid tank, a lysing liquid tank, a waste water tank, a detecting box, a primary filter and a secondary filter, and an outlet of the primary filter.
  • the dyeing agent inlet port is arranged on the detection box, and the sample pump, the flushing pump and the cracking pump are respectively arranged on the outlets of the sample box, the flushing liquid tank and the dyeing agent tank, and the outlet of the sample pump passes through the filtering valve and the second One port of the stage filter is connected, and the other port of the second stage filter is connected to the outlet of the flushing pump via a flushing valve, and the connecting pipe between the secondary filter and the flushing valve is connected to the waste water tank through the drain valve, and the liquid of the detecting box is connected.
  • the mouth inlet valve is connected with the connection line between the filter valve and the secondary filter, and the outlet of the cracking pump is connected to the detection box, and the detection box is provided with a fluorometer.
  • the primary filter described in the present invention is a nylon mesh having a mesh diagonal length of 35-50 um; the secondary filter is a microporous filter having an absolute pore diameter of 10 um; the fluorometer emits light at a wavelength of 460-490 nm.
  • the received excitation light has a wavelength of 510-550 nm.
  • the lower part of the detection box is connected to the waste water tank via a discharge valve.
  • the 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
  • the relative fluorescence intensity reference value only needs to be tested once, and it can become in multiple or even countless inspections.
  • the reference value has the advantages of simple structure, convenient use, high detection speed, high precision and reliable data.
  • Figure 1 is a schematic view showing the structure of a medium speed detecting device of the present invention.
  • Figure 2 is a graph showing the relationship between the relative fluorescence intensity and the viable cell content of Tetrahymena in the present invention. .
  • a 10-50um biological rapid detection method for ship ballast water comprises the following steps: a filter made of a nylon mesh having a mesh diagonal length of 50 um and a pressure to be discharged after being treated by a ballast water treatment device
  • the Tris-HCl backwashing small pore size filter, the trapped living organism with a size of 10-50 um is flushed into the sample tank; the live cell fluorescent stain fluorescein diacetate 20 mg is added to the sample tank, and staining is performed for 10 minutes.
  • cell lysate is a mixture of methanol and chloroform in a volume ratio of 1:2
  • the fluorometer is used for detection. The relative fluorescence intensity of the liquid in the sample cell.
  • the relative fluorescence strong reference value is obtained as follows: 60 known ballast water samples to be discharged containing 9 10-50 um surviving organisms per ml are selected as reference test samples; for each reference test sample, the following operations are performed: A filter made of a nylon mesh with a mesh length of 50 ⁇ m is used to filter out the living organism of not less than 50 ⁇ m in the reference test sample to obtain a filtrate; 200 ml of the filtrate is taken, and the living organism is less than 10 ⁇ m.
  • the average of the relative fluorescence intensities obtained after the treatment of the 60 reference test samples was calculated, which is the relative fluorescence intensity reference value. Comparing the relative fluorescence intensity value obtained after the ballast water to be treated with the relative fluorescence intensity reference value, it can be determined whether the ballast water sample to be tested meets the discharge standard.
  • the reference test sample is obtained by filtering the viable organism of not less than 50 um in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter to obtain a filtrate; taking Aml filtrate Use a small pore size filter to filter and intercept the living organisms of not less than 10um in the filtrate, and the living organisms less than 10um are filtered out with the liquid; backwash the small pore size filter with 3-10% Aml cleaning solution, the size will be intercepted
  • the 10-50um surviving organism is flushed into the sample tank; the liquid in the sample tank is detected by a FDA-CMFDA method under a fluorescence microscope, and when the content of the 10-50um surviving organism meets the requirements, it can be used as a reference test sample.
  • Cell lysate can leaching fluorescein inside living cells, improving the accuracy of the test results.
  • the FDA has two conjugated acetic acid free radicals. It is a non-polar substance that can pass freely through the algal cell membrane and is hydrolyzed to fluorescein by non-specific enzymes such as esterase, protease and lipase in the cell.
  • the FDA is a colorless compound that does not have fluorescence itself, and the reaction product fluorescein is a polar and fluorescent luminescent material that is chemically stable, is not easily decomposed, and is difficult to pass through the cell membrane and accumulate in cells.
  • the relationship between the relative fluorescence intensity and the biological content of 10-50 um was used to prepare different concentrations of tetraterpene solution, and the viable cell content was determined by FDA-CMFDA method. Each solution was added to the apparatus of the present invention and tested for relative fluorescence intensity using the method of the present invention. Taking the living cell content of tetradium algae as the x-axis and the measured relative fluorescence intensity as the y-axis, FIG. 2 is made. As can be seen from FIG. 2, the detection result of the present invention has a good one-to-one correspondence with the actual living cell content. It reflects the amount of viable cells in the sample.
  • a 10-50um biological rapid detection method for ship ballast water comprises the following steps:
  • the method for obtaining the relative fluorescence strong reference value is as follows:
  • the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer
  • each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained
  • the data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
  • A is any number, preferably a natural number from 100 to 500.
  • the reference test sample described in the step a in the present invention is obtained by filtering a surviving organism of not less than 50 ⁇ m in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter, The filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organisms of not less than 10 ⁇ m in the filtrate, and the living organisms less than 10 ⁇ m are filtered with the liquid; the cleaning solution of 3-10% Aml is used.
  • the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
  • the reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 ⁇ m in the filtrate, and a living organism of less than 10 ⁇ m with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Under the microscope, when the content of 10-50 um surviving organisms meets the requirements, the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
  • UV ultraviolet
  • the large-aperture filter is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; and the small-aperture filter is a microporous filter having an absolute pore diameter of 10 ⁇ m.
  • Membrane-made, backwashable filter The living cell fluorescent staining agent is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank.
  • the staining reaction time is 5-30 (10) minutes.
  • the PBS is: lead sulfide, and Tris-HCl is: tris(hydroxymethyl)aminomethane.
  • the cell lysate is one of a mixture of methanol and chloroform (1:1:3 by volume) or acetone.
  • FIG. 1 shows: including sample box 2, flushing liquid tank 13, cracking liquid tank 3, waste water tank 14, detecting tank 17, primary filter 1 and secondary filter 8, and primary filter 1 is using mesh diagonal
  • a secondary filter is a filter made of a microporous membrane with an absolute pore size of 10 um.
  • the outlet of the primary filter is connected to the inlet of the sample box 2, and the liquid filtered by the primary filter enters the connection line between the primary filter and the sample box 2 in the sample box 2, and may be provided with a primary pump or a first pump.
  • the stage filter is placed above the sample box 2 and naturally flows in by the gravity of the liquid.
  • the detection tank 17 is provided with a dye inlet port 12, and the sample tank 4, the flushing pump 13 and the cracking pump 5 are respectively disposed at the outlets of the sample tank 2, the flushing liquid tank 14 and the lysing tank 3, and the outlet of the sample pump 4 is filtered.
  • the valve 6 is connected to one port of the secondary filter 8, the other port of the secondary filter 8 is connected to the outlet of the flushing pump 13 via the flushing valve 11, and the connecting pipe between the secondary filter 8 and the flushing valve 11 is discharged through the draining valve 10 is connected to the waste water tank 15, and the liquid inlet of the detection tank 17 is connected to the connection line between the filter valve 6 and the secondary filter 8 via the inlet valve 9, and the outlet of the cracking pump 5 is connected to the detection tank 17 via the cracking valve 7.
  • the detection box 17 is provided with a fluorometer 18, and the detection probe of the fluorometer 18 is disposed in the detection box 17; the emission wavelength of the fluorometer is 460-490 nm, and the wavelength of the received excitation light is 510-550 nm; the lower part of the detection box
  • the discharge valve 16 is connected to the waste water tank 15.
  • the 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
  • the ballast water sample to be tested is placed in the primary filter, and the primary filter filters out the living organism of not less than 50um in the ballast water sample.
  • the filtered filtrate enters the sample box, and the cleaning liquid and the cell lysate are respectively added to the rinsing liquid tank and the lysing liquid tank; the filter valve and the liquid discharging valve are opened, the other valves are closed, the sample pump is operated, and the sample is quantitatively sampled.
  • the filtrate in the tank is filtered through a secondary filter, and the filtered liquid is discharged into the waste water tank.
  • the living organisms not less than 10um are trapped by the secondary filter; the filter valve and the drain valve are closed, and the dye valve and the dye valve are The discharge valve is also closed, the flush valve and the inlet valve are opened, the flushing pump is operated, the secondary filter is backwashed with a quantitative flushing liquid, and the 10-50 um of the trapped organism is flushed into the test chamber; the flushing valve is closed and Into the liquid inlet valve, add appropriate amount of live cell fluorescent stain to the sample box through the inlet of the staining agent, and fluorescently stain the living cells; after the living cells are dyed (about 10 minutes), the cracking valve is opened, and the pyrolysis pump works.
  • the relative fluorescence intensity reference value is only required to be taken once, and it can be used as a reference value in multiple or even countless tests.
  • the invention has the advantages of simple structure, convenient use, high detection speed, high precision and reliable and reliable data.

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Abstract

L'invention concerne un procédé et un dispositif de détection rapide d'organismes de 10 à 50 µm dans de l'eau de ballast de navire, se rapportant à des procédés et des dispositifs de détection pour eau de ballast, le procédé comprenant les étapes suivantes : filtration d'un échantillon d'eau de ballast à l'aide d'un filtre pour en éliminer par filtration les organismes vivants d'une taille supérieure à 50 µm et obtention d'un filtrat ; filtration à l'aide d'un filtre présentant une petite taille de maille et piégeage des organismes vivants d'une taille supérieure ou égale à 10 µm dans le filtrat ; lavage à contre-courant du filtre présentant une petit taille de maille à l'aide d'un fluide de nettoyage et évacuation des organismes vivants présentant une taille de 10 à 50 µm en direction d'une rainure de recueil d'échantillon ; addition d'un colorant fluorescent pour cellule vivante dans la rainure de recueil d'échantillon pour coloration ; addition d'une solution de lyse cellulaire dans la rainure de recueil d'échantillon ; détection de l'intensité de fluorescence relative du liquide présent dans la rainure de recueil d'échantillon à l'aide d'un fluoromètre ; et détermination pour savoir si la norme est atteinte par comparaison de l'intensité de fluorescence relative et de la valeur de référence, le dispositif de détection étant un système de détection automatique équipé d'un récipient, d'une pompe à liquide et d'un fluoromètre incorporés à l'intérieur de celui-ci.
PCT/CN2017/099893 2016-08-31 2017-08-31 Procédé et dispositif de détection rapide d'organismes de 10 à 50 μm dans de l'eau de ballast de navire WO2018041215A1 (fr)

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CN106244665A (zh) * 2016-08-31 2016-12-21 威海中远造船科技有限公司 船舶压载水中10‑50um生物快速检测方法及装置
CN107192655A (zh) * 2017-07-26 2017-09-22 连云港市质量技术综合检验检测中心 一种家用纯净水机隐孢子虫滤除率检验方法
CN108827891A (zh) * 2018-06-21 2018-11-16 上海海事大学 船舶压载水微藻细胞生物量检测系统及方法
CN109374584A (zh) * 2018-08-27 2019-02-22 九江精密测试技术研究所 一种船舶压载水生物有效性实时在线监测装置
CN111413479B (zh) * 2020-04-14 2023-03-21 诸暨市金桥实业有限公司 水质检测方法和系统

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