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
(*contributed equally to this work) 发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1779 浏览次数: 10460
评审: Valentine V TrotterLaura Molina-GarcíaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2015
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.
Materials and Reagents
- Balch tubes (Bellco Glass Inc., catalog number: 2048-00150 )
- Butyl Rubber Stoppers (Bellco Glass Inc., catalog number: 2048-11800A )
- 11 mm Aluminum Seal Crimps (Wheaton, catalog number: 224176-01 ) (Figure 1A)
- 20 mm Aluminum Seal Crimps (Wheaton, catalog number: 224178-01 )
- 18 G and 22 G BD PrecisionGlide Needle (Becton Dickinson, catalog number: 305195 )
- 22 G BD PrecisionGlide Needle (Becton Dickinson, catalog number: 305155 )
- Hamilton Gas Tight Syringe [1705 Sl 50 μl Syr (22s, 2”, 2) L] (Hamilton Company, catalog number: 80956 ) (Figure 1)
- Autosampler vials (National Scientific, catalog number: G4012-1W )** (Figure 1A)
- 11 mm straight plug stopper, natural red rubber (Wheaton, catalog number: 224100-030 )** (Figure 1A)
- UHP 99.99% Methane Gas Tank (Airgas, model: LW908 )
- Nalgene Labtop cooler (Sigma-Aldrich, catalog number: C2312-1EA )
- Nalgene Labtop cooler, Jr. (Sigma-Aldrich, catalog number: C2437-1 EA )
- 1 ml, 5 ml and 10 ml BD TB Syringes (Becton Dickinson, catalog number: 309624 , 309632 and 309640 )
- 15 ml sterile polypropylene Falcon conical centrifuge tubes (Corning, catalog number: 352196 )
- Sterile PVDF syringe filter (17 mm diameter, 0.2 μm pore size) (Thermo Fisher Scientific, catalog number: F25136 )
- 20 ml BD Luer Lok Disposable syringe (Becton Dickinson, catalog number: 302830 )
- 16 x 132 mm mm Type 1 glass A borosilicate glass test tubes (Bellco Glass Inc., catalog number: 2011-16125 )**
- 16 mm KAP-UTS test tube caps, various colors (Bellco Glass Inc., catalog number: 2007-16005 )**
- 1 ml micropipette (Mettler-Toledo, Rainin, model: L-1000 )*
- 200 μl micropipette (Mettler-Toledo, Rainin, model: L-200 )*
- 20 μl micropipette (Mettler-Toledo, Rainin, model: L-20 )*
- 20 μl HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-366 )*
- 200 μl HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-388 )*
- 1 ml HydroLogix SoftFit-L Pipet Tips (VWR International, catalog number: 89031-430 )*
- Envision paper towels (Georgia-Pacific, catalog number: 23504 )
- Hamilton gastight tapered syringe as injection needle (Hamilton Company, catalog number: 5181-8809 ) (Figure 1)
- UHP Air Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW700 )
- UHP Helium Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW800P )
- UHP Nitrogen Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: LW 411P )
- UHP Hydrogen Gas Tank with regulator plumbed to GC (Matheson Tri-Gas®, model: SG SPPULW500P )
- ddH2O
- Mupirocin (Sigma-Aldrich, catalog number: M7694 )**
- Sodium hydroxide (NaOH) anhydrous pellets (Sigma-Aldrich, catalog number: S8045-500 G )
- HS culture medium
- 3-N-(morpholino) propanesulfonate (MOPS) (pH 6.8)
- UHP 100% nitrogen (Matheson Tri-Gas®, model: SG SPPULW411 )
- 50 mM methanol
- General preparation of anaerobic solutions (see Recipes)
- 200x mupirocin stock (see Recipes)
- Plain medium (no C source) (see Recipes)
- 2x C medium (see Recipes)
Notes:- *Materials and reagents are only used for anaerobic condition.
- ** Materials and reagents can be used for both anaerobic and aerobic conditions.
- *Materials and reagents are only used for anaerobic condition.
Equipment
- 250 ml beaker (Thermo Fisher Scientific, catalog number: FB-100-250 )
- Anaerobic chamber or glove box with (Coylab, model: type B ) (Figure 2)
- Agilent 7890A Gas Chromatograph with Flame Ionization detector (Agilent Technologies, model: G3440A )
- Agilent Autosampler (Agilent Technologies, model: G4513A )
- Agilent Technologies OpenLAB CDS ChemStation Edition Rev C.01.02
- GS-CarbonPLOT GC Column (Agilent Technologies, catalog number: 113-3132 )
- Agilent Liner 4mm ID tap GW (Agilent Technologies, catalog number: 5062-3587 )
- Merlin Microseal High-Pressure Replacement Septum (Restek Corporation, catalog number: 22812 )
- IEC Medilite Microcentrifuge (Thermo Fisher Scientific, catalog number: 004480F ) (Figure 2)
Note: Equipment is used for anaerobic condition. - Spectronic 20D+ (Thermo Fisher Scientific, catalog number: 14-385-129 )
Software
- Agilent OpenLAB CDS ChemStation software
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
微生物学 > 微生物新陈代谢 > 其它化合物
生物化学 > 其它化合物 > 烷烃
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