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Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures.

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Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography
通过气相色谱法检测微生物的甲烷产生和消耗水平

微生物学 > 微生物新陈代谢 > 其它化合物
作者: Jared T. Aldridge*
Jared T. AldridgeAffiliation: Department of Biochemistry, Redox Biology Center, N200 Beadle Center, University of Nebraska-Lincoln, Lincoln, USA
Bio-protocol author page: a3037
Jennie L. Catlett*
Jennie L. CatlettAffiliation: Department of Biochemistry, Redox Biology Center, N200 Beadle Center, University of Nebraska-Lincoln, Lincoln, USA
Bio-protocol author page: a3038
Megan L. Smith
Megan L. SmithAffiliation: Department of Biochemistry, Redox Biology Center, N200 Beadle Center, University of Nebraska-Lincoln, Lincoln, USA
Bio-protocol author page: a3039
 and Nicole R. Buan
Nicole R. BuanAffiliation: Department of Biochemistry, Redox Biology Center, N200 Beadle Center, University of Nebraska-Lincoln, Lincoln, USA
For correspondence: nbuan@unl.edu
Bio-protocol author page: a3040
 (*共同第一作者)
Vol 6, Iss 7, 4/5/2016, 2036 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1779

[Abstract] Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures.

[Abstract] 甲烷是能量密集的燃料,但是也是比CO 2更加不利于环境25倍的温室气体。 甲烷可以通过蛇形化,化学方式通过Sabatier或Fisher-Tropsh化学,或通过微生物生物地生产(Berndt等人,1996; Horita和Berndt,1999; Dry,2002; Wolfe, Thauer,1998; Metcalf>等人,2002)。 甲烷生物是通过产生甲烷气体作为代谢副产物而生长的厌氧古生菌(Wolfe,1982; Thauer,1998)。 我们的实验室已经开发和优化了三种不同的气相色谱仪利用测定法来表征甲烷菌代谢(Catlett等人,2015)。 在这里我们描述的终点和动力学测定可用于测量甲烷产生甲烷生物或甲烷营养微生物的甲烷消耗。 该方案可用于测量甲烷产生或通过微生物纯培养物或通过富集培养物消耗。

Materials and Reagents

  1. Balch tubes (Bellco Glass Inc., catalog number: 2048-00150 )
  2. Butyl Rubber Stoppers (Bellco Glass Inc., catalog number: 2048-11800A )
  3. 11 mm Aluminum Seal Crimps (Wheaton, catalog number: 224176-01 ) (Figure 1A)
  4. 20 mm Aluminum Seal Crimps (Wheaton, catalog number: 224178-01 )
  5. 18 G and 22 G BD PrecisionGlide Needle (Becton Dickinson, catalog number: 305195 )
  6. 22 G BD PrecisionGlide Needle (Becton Dickinson, catalog number: 305155 )
  7. Hamilton Gas Tight Syringe [1705 Sl 50 μl Syr (22s, 2”, 2) L] (Hamilton Company, catalog number: 80956 ) (Figure 1)
  8. Autosampler vials (National Scientific, catalog number: G4012-1W )** (Figure 1A)
  9. 11 mm straight plug stopper, natural red rubber (Wheaton, catalog number: 224100-030 )** (Figure 1A)
  10. UHP 99.99% Methane Gas Tank (Airgas, model: LW908 )
  11. Nalgene Labtop cooler (Sigma-Aldrich, catalog number: C2312-1EA )
  12. Nalgene Labtop cooler, Jr. (Sigma-Aldrich, catalog number: C2437-1 EA )
  13. 1 ml, 5 ml and 10 ml BD TB Syringes (Becton Dickinson, catalog number: 309624 , 309632 and 309640 )
  14. 15 ml sterile polypropylene Falcon conical centrifuge tubes (Corning, catalog number: 352196 )
  15. Sterile PVDF syringe filter (17 mm diameter, 0.2 μm pore size) (Thermo Fisher Scientific, catalog number: F25136 )
  16. 20 ml BD Luer Lok Disposable syringe (Becton Dickinson, catalog number: 302830 )
  17. 16 x 132 mm mm Type 1 glass A borosilicate glass test tubes (Bellco Glass Inc., catalog number: 2011-16125 )**
  18. 16 mm KAP-UTS test tube caps, various colors (Bellco Glass Inc., catalog number: 2007-16005 )**
  19. 1 ml micropipette (Mettler-Toledo, Rainin, model: L-1000 )*
  20. 200 μl micropipette (Mettler-Toledo, Rainin, model: L-200 )*
  21. 20 μl micropipette (Mettler-Toledo, Rainin, model: L-20 )*
  22. 20 μl HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-366 )*
  23. 200 μl HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-388 )*
  24. 1 ml HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-430 )*
  25. Envision paper towels (Georgia-Pacific, catalog number: 23504 )
  26. Hamilton gastight tapered syringe as injection needle (Hamilton Company, catalog number: 5181-8809 ) (Figure 1)
  27. UHP Air Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW700 )
  28. UHP Helium Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW800P )
  29. UHP Nitrogen Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: LW 411P )
  30. UHP Hydrogen Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW500P )
  31. ddH2O
  32. Mupirocin (Sigma-Aldrich, catalog number: M7694 )**
  33. Sodium hydroxide (NaOH) anhydrous pellets (Sigma-Aldrich, catalog number: S8045-500 G )
  34. HS culture medium
  35. 3-N-(morpholino) propanesulfonate (MOPS) (pH 6.8)
  36. UHP 100% nitrogen (Matheson Tri-Gas®, model: SG SPPULW411 )
  37. 50 mM methanol
  38. General preparation of anaerobic solutions (see Recipes)
  39. 200x mupirocin stock (see Recipes)
  40. Plain medium (no C source) (see Recipes)
  41. 2x C medium (see Recipes)
    Notes:
    1. *Materials and reagents are only used for anaerobic condition.
    2. ** Materials and reagents can be used for both anaerobic and aerobic conditions.

Equipment

  1. 250 ml beaker (Thermo Fisher Scientific, catalog number: FB-100-250 )
  2. Anaerobic chamber or glove box with (Coylab, model: type B ) (Figure 2)
  3. Agilent 7890A Gas Chromatograph with Flame Ionization detector (Agilent Technologies, model: G3440A )
  4. Agilent Autosampler (Agilent Technologies, model: G4513A )
  5. Agilent Technologies OpenLAB CDS ChemStation Edition Rev C.01.02
  6. GS-CarbonPLOT GC Column (Agilent Technologies, catalog number: 113-3132 )
  7. Agilent Liner 4mm ID tap GW (Agilent Technologies, catalog number: 5062-3587 )
  8. Merlin Microseal High-Pressure Replacement Septum (Restek Corporation, catalog number: 22812 )
  9. IEC Medilite Microcentrifuge (Thermo Fisher Scientific, catalog number: 004480F ) (Figure 2)
    Note: Equipment is used for anaerobic condition.
  10. Spectronic 20D+ (Thermo Fisher Scientific, catalog number: 14-385-129 )

Software

  1. Agilent OpenLAB CDS ChemStation software

Procedure

  1. Preparing a standard curve
    1. Prep autosampler vials by flushing with air. Stopper, crimp and label vials (Figure 1A).
    2. Open gas valve and regulator of 99.99% methane gas tank. Regulator is fitted with a stem that allows a needle to fit into the tip (Figure 3). The stem is fitted with an air tight septa to not allow undesired methane gas to leak out of the tank.
    3. Using a 50 µl gastight Hamilton syringe (Reno, NV), insert the needle into the end of the regulator (Figure 3). Be sure that the stopcock is in the off position before inserting the needle. Using two fingers on either side of the needle, help guide needle in to prevent the needle from bending.
    4. Once the Hamilton syringe is inserted past the septa, open the stopcock and withdraw more than the desired amount of methane (i.e. If needing 50 µl of CH4, withdraw about 55 µl). Be careful not to allow plunger of the syringe to come out of syringe completely.
    5. Close the stopcock and withdraw the needle from end of the regulator (Figure 2B-2C).
    6. Open the stopcock and dispel gas from syringe to the desired volume.
    7. Allow gas to equilibrate to atmospheric pressure and then close the stopcock.
    8. Inject methane gas into stoppered/crimped autosampler vial.
    9. Run methane capture method on Agilent GC (details described below).
    10. Suggested volumes of 99.99% methane to inject into standard vials: 5 µl, 10 µl, 20 µl, 30 µl, 40 µl, and 50 µl (Figure 4).

      Table 1. Gas chromatograph “Methane” method settings
      Inlet
      Mode
      Splitless
      Purge flow
      60 ml/min at 0.75 min
      Heater
      250 °C
      Pressure
      31.529 psi
      Septum purge flow
      3 ml/min standard
      Column
      Column type
      GS CarbonPLOT
      Flow
      6.5 ml/min
      Pressure
      31.529 psi
      Ave. velocity
      85.265 cm/sec
      Constant flow
      Yes
      Post run
      7.3213 ml/min
      ALS injection volume
      2 µl
      Oven
      Temperature
      145 °C
      Hold time
      3 min
      FID detector
      Heater
      300 °C
      H2 flow
      30 ml/min
      Air flow
      400 ml/min
      Makeup flow (N2)
      25 ml/min
      Flame
      On

  2. Calculations
    1. Table S1. GC Methane Calculations guides the user in making a methane standard to determine the amount of moles of methane produced by a culture. Within the spreadsheet, the “Variables” tab contains constants and variables used in the calculation. The constants used conform to Beadle Center, University of Nebraska-Lincoln, but can be modified to accommodate other atmospheric and temperature conditions.
    2. A standard curve needs to be generated each time the FID detector is turned on. Once the standard has been created, export the following data to Microsoft Excel: Date and Time, Sample_Name, Vial #, Retention Time (min), Height, Area, and Injection_DataFileDirectory. Copy and paste the exported data into the attached spreadsheet’s “Standard Data” tab, making sure numbers do not transfer as text and everything is in the correct column. If using Agilent OpenLAB CDS ChemStation software, a report layout can be created that only includes these fields in the order of the spreadsheet.
    3. Once the “Standard Data” tab is filled out, fill out the “Constants” tab with injection volume, vial volume, and the volumes of methane gas added to each standard vial, corresponding to what was entered on the “Standards” tab. The spreadsheet will complete the calculations. The standard curve created by default is in Peak Area vs Methane (nmoles). It is important to note that this standard curve is the amount of methane injected into the GC (2 µl injected from each autosampler vial). This can be used in a direct comparison to 2 µl sampled in the same way elsewhere. The number of nmoles has not been multiplied by the dilution factor that would represent the number of moles in the entire autosampler vial. (For a 1.99 ml autosampler vial, the dilution factor is 995.)
    4. When using the standard curve created by the attached spreadsheet, data obtained directly from the GC can be compared. When completing the End Point Assay, the peak areas obtained from each sample can be directly plugged into the standard curve equation. In a Kinetic Assay, the volume needs to be adjusted to account for the vial volume displaced by the cell suspension (intact resuspended cells). Once the amount of methane detected by the GC is calculated, dilution factors must be used to calculate the total amount of methane in the headspace of the given culture.

  3. End point assay
    1. Grow pure or enrichment cultures in Balch tubes to the desired optical density. For example, for pure cultures of Methanosarcina acetivorans C2A grown at 35 °C in HS medium with 125 mM methanol as carbon and energy source with a 1:100 inoculum early stationary phase (OD600 = ~ 0.9) is reached between 50 and 75 h (Catlett et al., 2015).
    2. Prepare autosampler vials by flushing with air. Stopper, crimp, and label vials.
    3. In the fume hood, take culture tubes and insert an 18 G needle three quarters of the way through the blue butyl stopper (Figure 5A). Do not push the needle through the stopper completely.
    4. Using a gas tight Hamilton syringe, insert the syringe needle into the 18 G needle (Figure 5B). Be sure that the Hamilton syringe stopcock is closed at this time (Figure 2C). The 18 G needle is used as a guide to help puncture the butyl stopper with the Hamilton syringe needle while keeping the syringe needle straight.
    5. Push the Hamilton syringe needle through the butyl blue stopper until the end of the needle becomes visible in the headspace of the culture tube (Figure 5B). Once again, do not push the 18 G needle completely through the stopper.
    6. Open the stopcock of the Hamilton syringe. Quickly and steadily withdraw the Hamilton syringe plunger to the desired volume of headspace. (When using the standard curve described above, it is best to withdraw 50 µl of headspace from the culture.) Once at the desired volume of gas, quickly turn the stopcock of the Hamilton syringe, sealing the gas inside the syringe. Do not let the gas escape to equilibrate with atmospheric pressure (as in section A). Depending on the pressure in the growing culture, the plunger on the Hamilton syringe may continue to move as the gas expands in the syringe.
    7. Withdraw the Hamilton syringe and 18 G needle from the culture’s butyl stopper.
    8. With the Hamilton stopcock still closed, insert the Hamilton syringe needle into a stoppered and crimped autosampler vial.
    9. Open the stopcock and inject all the gas from the syringe into the autosampler vial.
    10. Close the stopcock and withdraw the Hamilton syringe from the autosampler vial.
    11. Once the headspace from all the cultures has been collected, place the vials in the autosampler.
    12. Run the “Methane” method on the GC (Table 1).

  4. Kinetic assay
    1. Before beginning, have the following materials and reagents completely anaerobic and ready in the chamber: Mupirocin stock (3.5 mg/ml) (or appropriate protein synthesis inhibitor depending on the susceptibility profile of the organisms you are assaying), sterile 16 mm test tubes, autosampler vials, autosampler stoppers and crimps, IEC Medilite Microcentrifuge. Prepare a stock of plain medium (no carbon source), and a second stock of growth medium with twice the concentration of carbon source (2x C medium). It is recommended to store these stocks in the anaerobic chamber.
    2. Grow cultures in Balch tubes to desired optical density (exponential phase OD600 = ~0.3-0.5). Record OD of the culture.
    3. Bring into the anaerobic chamber: Balch tube cultures in a 4 °C Nalgene Labtop Cooler, sterile labeled microcentrifuge tubes, autosampler crimper, liquid waste container, solid waste container, autosampler vial rack, and forceps.
    4. Measure the amount of plain medium and 2x C medium required for the experiment (Table 2). Add protein synthesis inhibitor to desired concentration. For methanogens, add mupirocin to 70 µM.
    5. Once items are brought into the chamber, gently resuspend any settled cells in the 10 ml culture. Withdraw 5 ml using a syringe and place in a sterile test tube. Withdraw the remaining 5 ml and place in a separate test tube.
    6. Spin down cell cultures in the centrifuge for 5 min at 1,228 x g.
    7. Decant supernatant and resuspend each pellet in 5 ml/test tube of plain medium. Use the syringe to disrupt the pellet and spin gently to avoid lysing cells.
    8. Spin the resuspended cells for 5 min in the centrifuge at 1,228 x g.
    9. As the centrifuge is running, place autosampler vials on an autosampler vial rack. Seven vials are required to assay one strain: five vials for sample replicates, a medium-only control (medium without cells) and a no substrate cells-only control (cells in plain medium only, no carbon source).
    10. For 500 µl cell suspensions, Add 250 µl 2x C medium to the five sample replicates and to the medium-only control. Add 250 µl of plain medium to both the medium-only and to the no-cells controls (Table 2).
    11. When the centrifuge has halted, immediately decant supernatant. Tap the test tube onto a piece of paper towel to remove all residual media.
    12. Use 2 ml plain medium to resuspend and combine pellets into one tube. This should be 2 ml total resuspension for each 10 ml Balch tube culture. Keep cold in a 4 °C Nalgene Labtop Cooler.
    13. Add 250 µl of cell suspension to the sample replicates and the cells-only control.
    14. Place 200 µl of leftover cell suspension into a labeled microcentrifuge tube. This will be used to measure protein concentration by Bradford assay.
    15. Crimp and label vials. Forceps can be helpful in placing stoppers and crimps onto each vial.
    16. Remove the vials from the chamber and place in a 35 °C incubator for 5 min before placing vials in the GC autosampler.
    17. Do six runs of a seven-vial sequence using the “Methane” method on Agilent GC (Table 1). Each vial should be run in order before repeating the sequence for the next measurement, resulting in 20-40 min for methane to accumulate in each vial between measurements.
    18. To measure protein concentration of the cell suspensions, spin down the 200 µl cell suspension saved in step 14 at 1,500 x g for 3 min. Remove supernatant and resuspend with 200 µl ddH2O. Lyse cells and perform a Bradford with Coomassie reagent and 2 mg BSA standard. Methanogen cells grown in HS medium are easily lysed by resuspension in ddH2O by osmotic shock, but organisms grown in low-osmolarity medium or that have cell walls may require boiling and/or freeze-thaw and vortexing or sonicating to fully lyse.
    19. Graph the methane peak area vs time for each sample (Figure 6). Use the slopes to calculate the amount of methane produced or consumed with time (Table S1.).

      Table 2. Kinetic assay medium volumes and controls

Representative data



Figure 1. Crimper, gastight autosampler vials and gas-tight Hamilton syringes. A. Crimpers are used to seal autosampler vials using aluminum crimps and rubber stoppers. Hamilton syringes showing open (B) and closed (C) luer fittings.


Figure 2. Dual-sided custom Coy anaerobic chamber showing Medlite clinical centrifuge (blue lid)


Figure 3. Methane gas tank fitted with a septa


Figure 4. Example standard curve


Figure 5. Method for preparing gas standards and sampling gas headspace. A. An 18 G needle is pushed into the stopper about three-quarters of the way through. This acts as a guide for the Hamilton needle to push through the stopper and not bend. B. A Hamilton syringe is inserted into an 18 G needle and pushed through the rest of the stopper. Once the end of the Hamilton syringe needle is through the end of the stopper (arrow), headspace can be extracted. Do not push the Hamilton syringe so far into the stopper that the 18 G needle is pushed through the stopper, as this will allow headspace gas to quickly escape.


Figure 6. Example kinetic assay results

Recipes

Notes:

  1. Preparing anaerobic solutions is a technical skill. For general instructions, please refer to Wolfe and Metcalf (2010).
  2. Some Methanosarcina species of methanogens can be grown in medium of different osmolarity (low-salt, LS, or high-salt, HS) culture medium (Sowers et al., 1993). If growing other methanogens or methanotrophs, use the appropriate medium recipe for the organism(s) of interest.
  1. 200x mupirocin stock
    1. Under anaerobic conditions, stopper and crimp an empty Balch tube. Autoclave to sterilize.
    2. Dissolve 35 mg mupirocin in 0.5 ml 1 M NaOH in a 15 ml Falcon tube.
    3. Complete volume to 10 ml with ddH2O. Final concentration of mupirocin in the 200x stock solution is 3.5 mg/ml (14 mM).
    4.  Using sterile technique, fit a 22 G needle to a sterile 17 mm diameter 0.2 µm pore size PVDF syringe filter using a plastic 20 ml syringe. Slowly push the solution through the filter, through the needle, and into the sterile anaerobic Balch tube.
    5. Use vacuum-vortex technique to make the mupirocin stock solution anaerobic (Wolfe and Metcalf, 2010).
    6. Store at 4 °C for up to a month.
  2. Plain medium (no C source)
    Follow the same culture medium recipe as usual, but omit the carbon source. For methanogens that cannot produce methane from CO2 such as Methanosarcina acetivorans, the normal HS medium is prepared. For methanogens and autotrophs that can fix CO2, carbonate, bicarbonate, and CO2 gas should also be eliminated from the recipe and replaced with a buffer at the appropriate pH. For example, for Methanosarcina species that can produce methane from CO2, the bicarbonate in the normal HS medium recipe is replaced with 50 mM 3-N-(morpholino) propanesulfonate (MOPS) (pH 6.8), and the medium is sparged and dispensed into Balch tubes under 100% nitrogen.
  3. 2x C medium
    Add twice the concentration of carbon source to Plain medium (no C source). For example, when assaying Methanosarcina grown on HS medium, if the desired final concentration in the assay is 50 mM methanol, add 100 mM methanol to Plain HS medium to make 2x C HS medium.

Acknowledgements

This material is based upon work supported by the National Science Foundation under Grants IOS-1449525 and MCB-1449014, by the Water Environment Research Foundation grant NTRY6R14, and by the Nebraska Center for Energy Sciences Cycle 8 award to N. Buan. M. Smith was supported by an American Society for Microbiology Undergraduate Research Fellowship and a Pepsi UCARE Fellowship. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funding sources. The authors declare no competing interests.

References

  1. Berndt, M. E., Allen, D. E. and Seyfried, W. E. (1996). Reduction of CO2 during serpentinization of olivine at 300 °C and 500 bar. Geology 24: 351-354.
  2. Catlett, J. L., Ortiz, A. M. and Buan, N. R. (2015). Rerouting cellular electron Flux to increase the rate of biological methane production. Appl Environ Microbiol 81(19): 6528-6537.
  3. Dry, M. E. (2002). The fischer-tropsch process: 1950-2000. Catal Today 71, 227-241.
  4. Horita, J. and Berndt, M. E. (1999). Abiogenic methane formation and isotopic fractionation under hydrothermal conditions. Science 285(5430): 1055-1057.
  5. Metcalf, W. W., Griffin, B. M., Cicchillo, R. M., Gao, J., Janga, S. C., Cooke, H. A., Circello, B. T., Evans, B. S., Martens-Habbena, W., Stahl, D. A. and van der Donk, W. A. (2012). Synthesis of methylphosphonic acid by marine microbes: a source for methane in the aerobic ocean. Science 337(6098): 1104-1107.
  6. Sowers, K. R., Boone, J. E. and Gunsalus, R. P. (1993). Disaggregation of methanosarcina spp. and growth as single cells at elevated osmolarity. Appl Environ Microbiol 59(11): 3832-3839.
  7. Thauer, R. K. (1998). Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture. Microbiology 144 (Pt 9): 2377-2406.
  8. Wolfe, R. S. (1982). Biochemistry of methanogenesis. Experientia 38 198-201.
  9. Wolfe, R. S. and Metcalf, W. W. (2010). A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes. Anaerobe 16(3): 216-219.

材料和试剂

  1. 巴克管(Bellco Glass Inc.,目录号:2048-00150)
  2. 丁基橡胶塞(Bellco Glass Inc.,目录号:2048-11800A)
  3. 11 mm铝密封卷边(Wheaton,目录号:224176-01)(图1A)
  4. 20 mm铝密封卷边(Wheaton,目录号:224178-01)
  5. 18 G和22 G BD PrecisionGlide针(Becton Dickinson,目录号:305195)
  6. 22G BD PrecisionGlide针(Becton Dickinson,目录号:305155)
  7. Hamilton气体注射器[1705 Sl50μlSyr(22s,2",2)L](Hamilton Company,目录号:80956)(图1)
  8. 自动进样器样品瓶(National Scientific,目录号:G4012-1W)**(图1A)
  9. 11毫米直塞塞,天然红橡胶(Wheaton,目录号:224100-030)**(图1A)
  10. UHP 99.99%甲烷气罐(Airgas,型号:LW908)
  11. Nalgene Labtop冷却器(Sigma-Aldrich,目录号:C2312-1EA)
  12. Nalgene Labtop冷却器(Sigma-Aldrich,目录号:C2437-1EA)
  13. 1ml,5ml和10ml BD TB注射器(Becton Dickinson,目录号:309624,309632和309640)
  14. 15ml无菌聚丙烯Falcon锥形离心管(Corning,目录号:352196)
  15. 无菌PVDF注射器过滤器(17mm直径,0.2μm孔径)(Thermo Fisher Scientific,目录号:F25136)
  16. 20ml BD Luer Lok一次性注射器(Becton Dickinson,目录号:302830)
  17. 16×132mm mm 1型玻璃硼硅酸盐玻璃试管(Bellco Glass Inc.,目录号:2011-16125)**
  18. 16mm KAP-UTS试管帽,各种颜色(Bellco Glass Inc.,目录号:2007-16005)**
  19. 1ml微量移液管(Mettler-Toledo,Rainin,型号:L-1000)*
  20. 200μl微量移液管(Mettler-Toledo,Rainin,型号:L-200)*
  21. 20μl微量移液管(Mettler-Toledo,Rainin,型号:L-20)*
  22. 20μlHydroLogix SoftFit-L移液管吸头(VWR International,目录号:89031-366)*
  23. 200μlHydroLogix SoftFit-L移液管吸头(VWR International,目录号:89031-388)*
  24. 1 ml HydroLogix SoftFit-L移液管吸头(VWR International,目录号:89031-430)*
  25. Envision纸巾(Georgia-Pacific,目录号:23504)
  26. 作为注射针的Hamilton气密锥形注射器(Hamilton Company,目录号:5181-8809)(图1)
  27. UHP空气罐,带有调节器连接到GC(Matheson Tri-Gas ?,型号:SG SPPULW700)
  28. UHP氦气罐,调节器连接到GC(Matheson Tri-Gas ,型号:SG SPPULW800P)
  29. UHP氮气罐,调节器连接到GC(Matheson Tri-Gas ,型号:LW 411P)
  30. UHP氢气罐,调节器连接到GC(Matheson Tri-Gas ,型号:SG SPPULW500P)
  31. ddH sub 2 O
  32. 莫匹罗星(Sigma-Aldrich,目录号:M7694)**
  33. 氢氧化钠(NaOH)无水粒料(Sigma-Aldrich,目录号:S8045-500G)
  34. HS培养基
  35. 3-N-(吗啉代)丙磺酸盐(MOPS)(pH6.8)
  36. UHP 100%氮气(Matheson Tri-Gas,型号:SG SPPULW411)
  37. 50 mM甲醇
  38. 厌氧溶液的一般准备(参见配方)
  39. 200x莫匹罗星储备(见配方)
  40. 普通介质(无C源)(参见配方)
  41. 2x C介质(见配方)
    注意:
    1. *材料和试剂仅用于厌氧条件。
    2. **材料和试剂可用于厌氧和有氧条件。

设备

  1. 250ml烧杯(Thermo Fisher Scientific,目录号:FB-100-250)
  2. 厌氧室或手套箱(Coylab,型号:B型)(图2)
  3. 带有火焰离子化检测器的Agilent 7890A气相色谱仪(Agilent Technologies,型号:G3440A)
  4. Agilent自动进样器(Agilent Technologies,型号:G4513A)
  5. Agilent Technologies OpenLAB CDS ChemStation版本C.01.02
  6. GS-CarbonPLOT GC柱(Agilent Technologies,目录号:113-3132)
  7. 安捷伦衬管4mm ID螺丝GW(安捷伦科技公司,目录号:5062-3587)
  8. Merlin Microseal高压替代隔垫(Restek Corporation,目录号:22812)
  9. IEC Medilite微量离心机(Thermo Fisher Scientific,目录号:004480F)(图2)
    注意:设备用于厌氧条件。
  10. Spectronic 20D +(Thermo Fisher Scientific,目录号:14-385-129)

软件

  1. Agilent OpenLAB CDS ChemStation软件

程序

  1. 准备标准曲线
    1. 通过用空气冲洗制备自动进样器小瓶。塞子,卷曲和标签瓶(图1A)。
    2. 打开燃气阀和调节器的99.99%甲烷气罐。调节器 装配有允许针安装到尖端中的杆(图3)。 阀杆配有气密隔膜,以防止不需要的 甲烷气体泄漏出水箱
    3. 使用50μl气密 Hamilton注射器(Reno,NV),将针插入末端 调节器(图3)。确保旋塞阀处于关闭位置 然后插入针。使用两个手指在两侧 针,帮助引导针以防止针弯曲
    4. 一旦汉密尔顿注射器插入隔垫,打开活塞 并且抽出超过所需量的甲烷(即如果需要50 ?μlCH 4,退出约55μl)。小心不要让柱塞 注射器完全从注射器中取出
    5. 关闭活栓并从调节器的末端取出针(图2B-2C)。
    6. 打开活塞并从注射器中排出所需体积的气体
    7. 让气体平衡到大气压,然后关闭活栓。
    8. 将甲烷气体注入带塞/卷曲的自动进样器样品瓶中
    9. 在Agilent GC上运行甲烷捕获方法(详情如下所述)。
    10. 注入标准瓶中的99.99%甲烷的建议体积:5μl,10μl,20μl,30μl,40μl和50μl(图4)。
      表1.气相色谱仪"甲烷"方法设置
      进入
      模式
      不分页
      清除流程
      60分钟/0.75分钟
      加热器
      250℃
      压力
      31.529 psi
      隔垫吹扫流量
      3 ml/min标准

      列类型
      GS CarbonPLOT
      流量
      6.5 ml/min
      压力
      31.529 psi
      Ave.速度
      85.265cm/sec
      恒定流量

      后运行
      7.3213ml/min
      ALS注入量
      2微升
      烤箱
      温度
      145℃
      保持时间
      3分钟
      FID检测器
      加热器
      300℃
      H <2> 流程
      30 ml/min
      气流
      400 ml/min
      化妆流量(N <2)
      25 ml/min
      火焰


  2. 计算
    1. 表S1。 GC甲烷计算指导用户制定甲烷标准 确定培养物产生的甲烷的摩尔量。中 电子表格,"变量"选项卡包含常量和变量 用于计算。使用的常数符合Beadle Center, 内布拉斯加大学林肯分校,但可以修改以容纳其他 ?大气和温度条件
    2. 每次FID检测器需要产生标准曲线 ?打开。创建标准后,导出以下内容 数据到Microsoft Excel:日期和时间,Sample_Name,Vial#,保留 时间(分钟),高度,区域和Injection_DataFileDirectory。复制和 将导出的数据粘贴到附加的电子表格的"标准数据" 标签,确保数字不会作为文本传输,一切都在 正确的列。如果使用Agilent OpenLAB CDS ChemStation软件,a ?可以创建只包括这些字段的报表布局 电子表格的顺序。
    3. 填写"标准数据"标签后 ?出来,用进样体积,样品瓶体积填充"常数"选项卡, 和加入每个标准小瓶的甲烷气体的体积, 对应于在"标准"选项卡上输入的内容。的 电子表格将完成计算。创建标准曲线 默认情况下为峰面积vs甲烷(nmoles)。重要的是要注意 该标准曲线是注入GC中的甲烷的量 (从每个自动进样器小瓶注射2μl)。这可以直接使用 ?与以相同方式在其他地方取样的2μl比较。的数量 nmoles没有乘以稀释因子 表示整个自动进样器样品瓶中的摩尔数。 (为一个 1.99 ml自动进样器样品瓶,稀释倍数为995.)
    4. 什么时候 使用由附加的电子表格创建的标准曲线,数据 直接从GC中获得可以进行比较。完成结束时 点测定,从每个样品获得的峰面积可以直接 插入标准曲线方程。在动力学测定中,体积 ?需要调整以考虑到被移位的瓶体积 细胞悬浮液(完全重悬细胞)。一次甲烷的量 检测由GC计算,稀释因子必须使用 计算给定顶空中甲烷的总量 文化。

  3. 终点测定
    1. 在Balch管中培养纯净或富集培养物至所需的 光密度。例如,对于甲烷八叠球菌的纯培养物 在含有125mM甲醇的HS培养基中35℃生长的乙酰化梭菌C2A 碳和能源与1:100接种早期固定相 (OD 600 =?0.9)在50和75小时之间达到(Catlett等人,2015)。
    2. 通过用空气冲洗制备自动进样器小瓶。塞子,卷曲和标签瓶。
    3. 在通风橱中,取培养管和插入18 G针三 ?四分之一通过蓝色丁基塞(图5A)。不要 将针完全推入塞子。
    4. 使用气密 ?Hamilton注射器,将注射器针头插入18 G针 (图5B)。确保Hamilton注射器活塞在关闭 这一次(图2C)。 18 G针用作指导来帮助 用Hamilton注射器针穿刺丁基塞子 保持注射器针直。
    5. 推入Hamilton注射器 针通过丁基蓝塞子直到针的末端 变得在培养管的顶部空间中可见(图5B)。一旦 再次,不要将18 G针完全穿过塞子
    6. 打开Hamilton注射器的活塞。快速和稳定 撤回Hamilton注射器柱塞至所需体积 顶空。 (使用上述标准曲线时,最好 ?从培养物中取出50μl的顶空) 体积的气体,迅速转动Hamilton注射器的活塞, 密封注射器内的气体。不要让气体逃逸 用大气压平衡(如A节)。取决于 在生长培养中的压力,柱塞在Hamilton注射器上 ?可能会随着气体在注射器中膨胀而继续移动
    7. 从文化的丁基塞子取出Hamilton注射器和18 G针。
    8. 在Hamilton止回阀仍然关闭的情况下,将Hamilton注射器针头插入带塞和卷曲的自动进样器样品瓶中。
    9. 打开活栓,将注射器中的所有气体注入自动进样器样品瓶。
    10. 关闭活栓,从自动进样器样品瓶中取出Hamilton注射器
    11. 一旦收集到所有培养物的顶空,将样品瓶放入自动进样器中。
    12. 在GC上运行"甲烷"方法(表1)。

  4. 动力学测定
    1. 开始之前,请准备以下材料和试剂 完全厌氧,准备在腔室:莫匹罗星股票(3.5 mg/ml)(或合适的蛋白质合成抑制剂, 您正在测定的生物体的磁敏度曲线),无菌16 mm ?试管,自动进样器样品瓶,自动进样器塞子和卷曲,IEC Medilite微量离心机。准备普通介质(不含碳 来源),和生长培养基的第二股两倍 碳源浓度(2×C培养基)。建议存储 ?这些储存在厌氧室中
    2. 在Balch管中培养培养物至所需的光密度(指数期OD 600 =?0.3-0.5)。记录培养物的OD
    3. 进入厌氧室:Balch管培养在4℃ Nalgene Labtop冷却器,无菌标记的微量离心管, 自动进样器卷曲机,液体废物容器,固体废物容器, 自动进样器样品架和镊子
    4. 测量平原的量 培养基和2xC培养基(表2)。加 蛋白合成抑制剂至所需浓度。对于产甲烷菌, 将莫匹罗星添加至70μM
    5. 一旦物品被带入腔室中, 轻轻地重悬在10ml培养物中的任何沉降的细胞。提取5 ml 使用注射器并置于无菌试管中。撤销剩余的 ?5 ml,并置于单独的试管中
    6. 在离心机中以1228×g离心细胞培养物5分钟。
    7. 滗析上清液并将每个沉淀重悬于5ml /试管中 普通介质。使用注射器破碎沉淀并轻轻旋转 避免溶解细胞
    8. 在离心机中以1,228×g旋转重悬的细胞5分钟。
    9. 当离心机运行时,将自动进样器样品瓶放在上 自动进样器样品架。需要七个小瓶来测定一种菌株: 用于样品重复的五个小瓶,仅培养基对照(培养基无 细胞)和无底物细胞对照(细胞在纯培养基中 只有,没有碳源)
    10. 对于500μl细胞悬浮液,加入250μl ?2x C培养基至五个样品重复和仅培养基 控制。添加250微升的普通培养基到中等和只 无细胞对照(表2)
    11. 当离心机停止时, 立即倾析上清液。点击试管到一张纸上 毛巾清除所有残留的介质。
    12. 使用2ml普通培养基 重悬并将丸粒合并成一个管。这应该是2毫升总 每个10ml Balch管培养物重悬。保持在4°C冷 Nalgene Labtop Cooler。
    13. 添加250微升的细胞悬浮液的样品重复和单元格的控制
    14. 将200μl剩余的细胞悬浮液放入标记 微量离心管。这将用于测量蛋白质浓度 ?通过Bradford测定
    15. 压接和标签小瓶。镊子有助于将塞子和卷曲放置在每个小瓶上。
    16. 从小室中取出小瓶,并置于35°C孵育器中5分钟,然后将小瓶放入GC自动进样器。
    17. 使用"甲烷"方法进行七个小瓶序列的六次运行 Agilent GC(表1)。在重复之前,每个小瓶应按顺序运行 用于下一次测量的序列,导致20-40分钟 甲烷在测量之间积累在每个小瓶中
    18. 至 测量细胞悬浮液的蛋白质浓度,自旋下降200 ?μl细胞悬液在1500×g下在步骤14中保存3分钟。去掉 上清液并用200μlddH 2 O重悬。溶解细胞并执行 Bradford,用考马斯试剂和2mg BSA标准。甲醇原细胞 在HS培养基中生长的细胞容易通过渗透在ddH 2 O 2中重悬浮而溶解 休克,但生物体生长在低渗透压介质或有细胞 壁可能需要煮沸和/或冻融和涡旋或声波处理 ?完全溶解。
    19. 绘制每个的甲烷峰面积对时间 样品(图6)。使用斜率计算甲烷的量 生产或消耗的时间(表S1。)。

      表2.动力学测定培养基体积和对照

代表数据



图1.压接器,气密性自动进样器样品瓶和气密的Hamilton注射器。A.压接器用于使用铝卷和橡皮塞密封自动进样器样品瓶。 Hamilton注射器显示开放(B)和关闭(C)路厄配件

图2.双面定制Coy厌氧室显示Medlite临床离心机(蓝色盖)


图3.装有隔垫的甲烷气罐


图4.示例标准曲线


图5.制备气体标准品和取样气体上部空间的方法。A.将18 G针推入塞子约四分之三的距离。这用作引导Hamilton针穿过塞子而不弯曲的引导。 B.将Hamilton注射器插入18G针中,并推动通过其余的塞子。一旦汉密尔顿注射器针的端部通过塞子的末端(箭头),可以提取顶部空间。不要将Hamilton注射器推入塞子,18 G针通过塞子,因为这将允许顶部气体快速逸出。


图6.实例动力学测定结果

食谱

注意:

  1. 准备厌氧溶液是一项技术技能。有关一般说明,请参阅Wolfe和Metcalf(2010)。
  2. 一些甲烷八叠球菌的甲烷八叠球菌种可在培养基中生长 不同的渗透压(低盐,LS或高盐,HS)培养基 (Sowers等人,1993)。如果生长其他产甲烷菌或甲烷营养菌, 使用感兴趣的生物体的适当的培养基配方。
  1. 200x莫匹罗星股票
    1. 在厌氧条件下,塞子并卷曲空的Balch管。高压灭菌。
    2. 将35mg莫匹罗星溶解在0.5ml 1M NaOH中的15ml Falcon管中
    3. 用ddH 2 O完全体积至10ml。莫匹罗星在200x储备溶液中的最终浓度为3.5mg/ml(14mM)
    4.  使用无菌技术,将22 G针头安装到无菌的17 mm直径 0.2μm孔径PVDF注射器过滤器,使用塑料20ml注射器。 慢慢推动溶液通过过滤器,通过针,和 进入无菌厌氧Balch管
    5. 使用真空涡流技术使莫匹罗星储备液厌氧(Wolfe和Metcalf,2010)。
    6. 储存于4°C长达一个月。
  2. 普通介质(无C源)
    遵循与往常一样的培养基配方,但省略碳源。对于不能从CO 2产生甲烷的产甲烷菌如乙酸甲烷八叠球菌,制备正常的HS培养基。对于可以固定CO 2的产甲烷菌和自养生物,碳酸盐,碳酸氢盐和CO 2气体也应当从配方中消除并且在适当的pH下用缓冲液替换。例如,对于可以从CO 2产生甲烷的甲烷八叠球菌物种,正常HS培养基配方中的碳酸氢盐用50mM 3-N-(吗啉代)丙磺酸盐( MOPS)(pH6.8)中,并在100%氮气下将培养基喷射并分配到Balch管中
  3. 2x C介质
    将两倍浓度的碳源加入纯培养基(无C源)。例如,当测定在HS培养基上生长的甲烷八叠球菌时,如果测定中所需的最终浓度是50mM甲醇,则向Plain HS培养基中加入100mM甲醇以制备2x C HS培养基。

致谢

该材料基于国家科学基金会授予的IOS-1449525和MCB-1449014,由水环境研究基金会授予NTRY6R14支持的工作,以及内布拉斯加州能源科学周期8中心授予N. Buan。 M.史密斯由美国微生物学会本科研究奖学金和百事可乐结盟奖。在本材料中表达的任何意见,发现,结论或建议是作者的,不一定反映资金来源的意见。作者声明没有竞争的利益。

参考文献

  1. Berndt,M.E.,Allen,D.E。和Seyfried,W.E。(1996)。 在300°C下橄榄石蛇纹石化期间还原CO 和500巴。 地质 24:351-354。
  2. Catlett,J.L.,Ortiz,A.M.and Buan,N.R。(2015)。 重新发送细胞电子通量以提高生物甲烷产生的速率。应用Environ Microbiol 81(19):6528-6537
  3. Dry,M.E。(2002)。 费 - 托过程:1950-2000

    111,227-241。
  4. Horita,J。和Berndt,M.E。(1999)。 在水热条件下的无生物甲烷形成和同位素分馏 科学 285(5430):1055-1057
  5. Metcalf,WW,Griffin,BM,Cicchillo,RM,Gao,J.,Janga,SC,Cooke,HA,Circello,BT,Evans,BS,Martens-Habbena,W.,Stahl,DAand van der Donk, 2012)。 海洋微生物合成甲基膦酸:有氧海洋中甲烷的来源。 337(6098):1104-1107。
  6. Sowers,K.R.,Boone,J.E。和Gunsalus,R.P。(1993)。 分解甲烷八叠球菌并在升高的摩尔渗透压浓度下作为单细胞生长。 Appl Environ Microbiol 59(11):3832-3839。
  7. Thauer,R.K。(1998)。 甲烷生成的生物化学:对Marjory Stephenson的致敬。 1998 Marjory Stephenson Prize Lecture。 Microbiology 144(Pt 9):2377-2406。
  8. Wolfe,R.S.(1982)。 产甲烷生物化学。 Experientia 38 198-201。
  9. Wolfe,R.S.and Metcalf,W.W。(2010)。 用于制备缺氧溶液或液体培养基的真空涡流技术,用于培养产甲烷菌或 其他严格的厌氧菌。 16(3):216-219。
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How to cite this protocol: Aldridge, J. T., Catlett, J. L., Smith, M. L. and Buan, N. R. (2016). Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography. Bio-protocol 6(7): e1779. DOI: 10.21769/BioProtoc.1779; Full Text



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(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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