High-purity methane (99.999%) is then added back to create an internal vessel pressure of 0 psig. A vacuum is then applied to create a partial vacuum in the gas-tight vessel (typically -15 psig).
Plates are streaked and placed in the jar under ambient atmospheric conditions. In these systems, vessels initially designed for maintenance of anaerobic cultures (e.g., Oxoid jars) are used. Typically for growth of methanotrophs on plates, a simple gassing system is used to provide methane.
Growth of methanotrophs can be challenging due to the necessity of having gas-tight systems in which methane can be easily provided to promote growth. ( 2002), and of haloalkaliphilic methanotrophs, as described by Kaluzhnaya et al., ( 2001).
Growth of halophilic methanotrophs as described by Heyer, et al ( 2005), Horz, et al. Growth of psychrophilic methanotrophs as described by Bowman, et al. Growth of acidophilic/thermoacidophilic methanotrophs as described by Dedysh, et al. These strains require different growth conditions, and specific media have been developed for these strains. Media for Growth of “Extreme” MethanotrophsĪs noted above, the physiological diversity of methanotrophs is quite broad, they can be isolated from many intriguing locations, including acidic bogs and hot springs, mud volcanoes, hypersaline lakes, etc. Mix the amounts designated above in 900 mL of H 2O, adjust to pH 7.0, and add H 2O to a final volume of 1L. Recipe for BTZ Growth Medium Ammonium Liquid Medium (BTZ)** One such example is BTZ, a growth medium ( Sharpe et al., 2007) designed for Methylomonas species (a genus within the ?-Proteobacteria) although one could conceivably use this for any mesophilic methanotroph: Many alternative growth media have been designed for methanotrophs. Alternative media for growth of mesophilic methanotrophs. This is another variation on NMS medium, in which both ammonium (0.25 g NH 4Cl) and nitrate (0.25 g KNO 3) are added, with the rest of the medium preparation identical to that for NMS as described above.ģ.1.4. Ammonium Nitrate Mineral Salts Medium (ANMS). The rest of the recipe and steps of preparation are identical to NMS medium.ģ.1.3. This medium is less common in practice than NMS, as some methanotrophs do not grow well with ammonium as a nitrogen source. Another common medium used for methanotrophic growth is “Ammonium Mineral Salts” medium (AMS) in which 0.5 g NH 4Cl is substituted for the 1 g of KNO 3 ( Whittenbury et al., 1970). This protocol is not recommended, as it is more likely that precipitates will form after autoclaving, which may complicate the maintenance of methanotrophic cultures.ģ.1.2. to achieve the final desired phosphate concentration). 
7(H 2O) in conjunction with Fe-EDTA, KNO 3, etc. Also, some labs add the components of the phosphate buffer directly to the base NMS recipe and then autoclave (i.e., the addition of 0.26 g KH 2PO 4 and 0.62 Na 2HPO 4 Vitamins often stimulate growth of methanotrophs. Various labs have different variations of NMS medium, with some omitting vitamins to decrease risk of contamination. It is important that the vitamin and Fe-EDTA solutions not be exposed to light to prevent photodegradation (wrapping stock bottles in aluminum foil is sufficient). 4(H 2O) 4 solutions are typically made at 1X and stored at 4☌. NOTE: It is IMPORTANT that copper be added AFTER autoclaving via a filter-sterilized 10 mM stock solution to reduce the probability of forming copper precipitates. The amount noted above (10 µM) usually allows good growth. It is common to vary the copper concentration from 0 (no additional copper) to 20 µM, although in some experiments as much as 100 µM has been added. Copper is typically added after autoclaving the base components using a stock solution of filter-sterilized CuCl 2. It is often of interest to vary the copper concentration as it is well-known that copper has a strong effect on methanotrophic activity, e.g., the canonical “copper-switch” ( Semrau et al., 2013 Stanley et al., 1983). If this is the case, deionized water can be used for medium preparation. It is preferable to use the purest water source available for growth of methanotrophs, e.g., deionized-distilled water, BUT on occasion methanotrophic growth can be poor in such water.
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