Cultivation techniques

The methanogens are strictly anaerobic prokaryotes that thrive only under reducing conditions. While many fermenters and sulphate reducers can cope with short time oxygen exposure, methanogens are the most oxygen-sensitive microorganisms

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known. This sensitivity requires a range of precautions to prevent oxygen exposure and also the use of specifically designed anoxic, reducing media.

2.3.1. Preparation of methanogen cultivation media

Methanogen media was prepared under anoxic conditions using a glass vessel (Figure 2.0.2.) based on the design of Widdel (1980). Basal mineral salts (Table 2.0.1.), SL10 trace element solution (Table 2.0.2.), and selenite-tungstate solution (Table 2.0.3.) were dissolved in reverse osmosis water and autoclaved within the media vessel for 1 hour at 121 °C. During autoclaving the tube leading to the glass bell was closed using a steel clip to prevent media loss. At least one of the screw caps of the side ports on top of the vessel was loosened during autoclaving to allow pressure exchange between the vessel interior and exterior. After autoclaving the media was removed from the autoclave at temperatures between 75 and 80°C and connected to a gas line. This was done to take advantage of the fact that the freshly boiled media is almost gas free, and therefore oxygen free. The incoming gas was sterilised by passing through a glass wool or cotton filter. After the vessel was connected the gas was kept flowing for about 5 minutes with one or both side ports partly opened to allow replacement of the headspace air in the vessel with oxygen-free gas, N2/CO2 or H2/CO2 (for carbonate buffered media; 80:20 v/v, 5 kPa) or N2 (for phosphate buffered media; 5 kPa) headspace. The media becomes saturated with the gas present in the headspace whilst cooling. Temperature-sensitive (e.g. vitamins, iron (II) chloride) or volatile (e.g.

Table 2.0.1. Mineral salts composition of FeS reduced methanogen cultivation media.

Mineral salt Solution concentration Media salinity

Freshwater Brackish Seawater

NaCl - 0.2 g 2 g 24.3 g

MgCl2 * 6 H2O - 0.25 g 0.7 g 10 g

CaCl2 * 2 H2O - 0.1 g 0.2 g 1.5 g

KCl - 0.1 g 0.2 g 0.66 g

NH4Cl 0.4 M 1 ml 1 ml 1 ml

KH2PO4 0.04 M 1 ml 1 ml 1 ml

NaBr 0.84 M - 0.1 ml 1 ml

NaF 0.07 M - 0.1 ml 1 ml

SrCl2 0.15 M - 0.1 ml 1 ml

H3BO3 0.4 M - 0.1 ml 1 ml

SL10* - 1 ml 1 ml 1 ml

SeWo^† - 0.2 ml 0.2 ml 0.2 ml

* Unchelated trace element solution (Widdel et al. 1983).

† Selenite-tungstate solution (Widdel and Bak 1992).

N.B. Addition mass/volumes are per litre

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bicarbonate, sulphide) media components were added to the cold media from sterile stock solutions via the side ports of the media vessel. Contamination of the media with airborne microbes is extremely unlikely due to the counter-flow of sterile headspace gas. The pH of the media was adjusted to 7.2-7.4 using autoclaved 1M NaOH or HCl if necessary. Media was dispensed under an autoclaved glass bell (to maintain sterility) into autoclaved screw top bottles for storage.

2.3.1.1. Buffering of methanogen media

The standard buffer used for methanogen media was a mixture of carbon dioxide and bicarbonate, which is also the natural buffer system in most aquatic and sedimentary systems. While carbon dioxide is added via the gas headspace, bicarbonate stock solutions are prepared alongside the media. For one litre of media 30 ml of a 1 M NaHCO3 solution was prepared and filled into a screw-cap bottle, ensuring that the bottle was only filled to a maximum of 60%. The screw cap with a rubber septum was tightly closed to prevent a loss of carbon dioxide during autoclaving. For safety reasons, the bottle with the bicarbonate solution is autoclaved within a plastic container, and is removed once the solution has reached room temperature.

Figure 2.0.2. Schematic diagram of media vessel used to prepare and dispense FeS-reduced mineral salts media.

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2.3.1.2. Reducing agents

Reducing agents are added to scavenge any oxygen that may still be present in the media after autoclaving or that diffuses in during incubation, and also to lower the redox potential of the media. The most common reducing agents used in microbiology are sodium dithionite (which is very selective for sulphate reducers) or and sodium sulphide. Iron monosulphide (FeS) was used in this study rather than sodium sulphide.

Iron monosulphide offers a number of advantages compared with sodium sulphide: (i) the added iron represent additional oxygen scavenging potential and iron reacts faster with oxygen than sulphide, (ii) as most of the sulphide is present in FeS particles, the free sulphide concentration is lower than in standard sulphide-reduced media, (iii) because during oxidation the colour changes from black to orange (iron oxohydroxide), there is no need for the addition of a redox indicator (resazurin) which may be inhibitory for some microorganisms, (iv) FeS particles offer surfaces for attachment. FeS is particulate, therefore accurate addition is very difficult. For this reason, iron (II) and sulphide were added separately from sterile stock solutions (see 2.3.1.3.). As iron (II) chloride solution is very acidic, and the sodium sulphide solution is very alkaline, it is necessary to let the media pH stabilise for 15 minutes after addition of these compounds before testing the media pH.

2.3.1.3. Media additions

The composition of the different media supplements are given in Tables 2.0.2. - 2.0.5.

The vitamin solution is sterile-filtered and the glass bottles wrapped with aluminium foil to prevent photodegradation. The vitamin solution was stored at 4°C. The trace metal solution is prepared using HCl as most metals would precipitate as metal oxides at neutral pH. The use of complexation agents, such as EDTA, was avoided as they may be inhibitory to methanogens. Selenite and tungstate are only soluble under alkaline conditions, and therefore cannot be added to the trace element solution. The trace element and selenite-tungstate solution are autoclaved, and can be stored at room temperature.

36 Table 2.0.2. Composition of SL10 trace element solution (Widdel et al. 1983)

Compound mg l^-1 (25%) and mix well. Add the above compounds and distilled water to make up to 1000 ml total volume, and mix well. Decant into screw top bottles and autoclave at 120 °C for 30 minutes.

Table 2.0.3. Composition of selenite and tungstate (SeWo) solution (Widdel and Bak 1992)

Compound mg l^-1

NaOH 400

Na2SeO3.5H2O 6

Na2WO3.2H2O 8

To prepare solution add the above components to 1000 ml of distilled water, and mix well.

Decant into screw top bottles and autoclave at 120 °C for 30 minutes.

Table 2.0.4. Temperature sensitive additions to methanogen cultivation media Media additions Solution concentration ml l^-1 of media

NaHCO3 solution 1 M 30

10 vitamin solution* - 2

Na2S^a 1 M 1.2

FeCl2b 1 M in 0.1 M HCl 0.5

* Composition of 10 vitamin solution (Balch et al. 1979) shown in Table 2.0.5.

a To prepare Na2S solution add distilled water to serum vial and sparge with N2

for 15 minutes. Add Na2S.9H2O using plastic tweezers, and flush vial headspace with N2. Seal vial with butyl rubber stopper and crimp. The resulting solution has a pH of around 14, and is therefore considered sterile.

b To prepare to FeCl2 solution add 0.1 M HCl into a glass vial with magnetic follower and gas with N2 for 15 minutes. Add FeCl2.4H2O and mix, whilst flushing vial with N2. Seal vial with butyl rubber stopper and crimp. Autoclave for 30 minutes at 120 °C.

Table 2.0.5. Composition of 10 vitamin solution (Balch et al. 1979)

Compound mg l^-1

To prepare the 10 vitamin solution add the above compounds to 1000 ml of distilled water and mix well. Filter sterilize the resulting solution into autoclaved screw cap bottles covered in foil.

Store at 4°C.

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2.3.2. Preparation of methanogen cultivation tubes

10 ml of media was transferred from screw cap bottles into 20 ml anaerobic tubes (Bellco, New Jersey, USA) under an N2/CO2 or N2 gas stream using a sterile glass syringe. Methanogen cultivation tubes were flushed with either N2/CO2 (80:20 v/v, 150 kPa) or N2 (150 kPa) using a gassing cannula (Figure 2.0.4.) for 1 minute prior to the transfer of media, and for 30 seconds following the transfer of media. Once media transfer and headspace flushing was complete the tubes were sealed with a butyl rubber bung and aluminium crimp.