2. M ATERIALS AND M ETHODS
2.3. Microbiological methods
2.3.1. Growth media and plates for cultivation and cloning
2.3.1.2. Liquid media and media plates
2.3.1.2.1. DE/NO2 medium used for the isolation of denitrifiers
Mineral salts DE-B (2.3.1.1.4) 5 ml
Trace elements DE (2.3.1.1.6) 5 ml
Agar 7.5 g
Mineral salts DE-A (2.3.1.1.3) ad 490 ml
Carbon sources (2.3.1.1.11) 6 ml
Vitamins DE (2.3.1.1.8) 1 ml
Nitrite (2.3.1.1.12) 3 ml
For the DE/NO2-medium used for the isolation of denitrifiers (2.3.2.1), the medium (modified from Atlas & Parks [2000]) was prepared in serum vials flushed with 100 % N2
before, during, and after the application of the components to gain anoxic conditions. The pH was adjusted to 6.8 - 7.0, and the solution was autoclaved. After cooling down to approximately 70 °C, carbon sources, vitamins, and nitrite were added with syringes that were flushed with sterile N2 (100 %) and mixed carefully to avoid bubbles in the medium. The medium was poured into sterile plastic Petri dishes in the oxygen-free chamber (100 % N2).
After solidification, the dishes were stored at room temperature in the oxygen-free chamber for at least 2 days before further use.
2.3.1.2.2. DE/N2O-medium used for the isolation of denitrifiers
Mineral salts DE-B (2.3.1.1.4) 5 ml
Trace elements DE (2.3.1.1.6) 5 ml
Agar 7.5 g
Mineral salts DE-A (2.3.1.1.3) ad 496 ml
Carbon sources (2.3.1.1.11) 3 ml
Vitamins DE (2.3.1.1.8) 1 ml
Nitrite (2.3.1.1.12) 0.1 ml
For the DE/N2O-medium used for the isolation of denitrifiers (2.3.2.1), the medium (modified from Atlas & Parks [2000]) was prepared in serum vials flushed with 100 % N2 before, during, and after the application of the components to gain anoxic conditions. The pH was adjusted to 6.8 - 7.0, and the solution was autoclaved. After cooling down to approximately 70 °C, carbon sources, vitamins, and nitrite were added with syringes that were flushed with sterile N2 (100 %) and mixed carefully to avoid bubbles in the medium. The medium was poured into sterile plastic Petri dishes in the oxygen-free chamber (100 % N2).
After solidification, the dishes were stored at room temperature in the oxygen-free chamber for at least 2 days before further use.
2.3.1.2.3. DE-plates to maintain isolates under oxic conditions
Yeast 0.5 g
Glucose 0.5 g
Agar 7.5 g
Mineral salts DE-B (2.3.1.1.4) 5 ml
Trace elements DE (2.3.1.1.6) 5 ml
Oxic mineral salts DE-A (2.3.1.1.3) ad 493 ml
Carbon sources (2.3.1.1.11) 6 ml
Vitamins DE (2.3.1.1.8) 1 ml
Plates of the DE-medium were used to gain plate colonies for the growth experiments of potential denitrifiers (2.3.2.2). The medium (modified from Atlas & Parks [2000]) was prepared with mineral salt solution DE-A that was prepared with normal, i.e., oxic instead of anoxic ddH2O. The pH was adjusted to 6.8 - 7.0, and the solution was autoclaved. After cooling down to approximately 70 °C, carbon sources and vitamins were added. The medium was poured into sterile plastic Petri dishes. After solidification, the dishes were stored at room temperature to get oxic conditions.
2.3.1.2.4. DE/ISO-medium for growth experiments with isolates
Yeast 0.06 g
Glucose 0.09 g
Mineral salts DE-B (2.3.1.1.4) 1 ml
Trace elements DE (2.3.1.1.6) 1 ml
Mineral salts DE-A (2.3.1.1.4) ad 100 ml
The DE/ISO-medium was used for growth experiments under oxic and anoxic conditions (2.3.2.2). The anoxic medium (modified from Atlas & Parks [2000]) used to analyze growth under anoxic conditions (2.3.2.2.2) was prepared in serum vials flushed with 100 % N2 before, during, and after the application of the components to gain anoxic conditions. The pH was adjusted to 6.8 - 7.0, and the solution was autoclaved. After cooling down, 14 ml medium was transferred into sterile anoxic tubes (butyl rubber stopped aluminium crimp sealed glass tubes; 24 ml) that were flushed with sterile 100 % N2 before and during the procedure. For each anoxic tube, 1.6 ml of combined C-sources and vitamins (2.3.1.1.14) was added.
The oxic medium used to analyze growth under oxic conditions (2.3.2.2.1) was prepared with mineral salt solution DE-A that was prepared with normal, i.e., oxic instead of anoxic ddH2O. The medium was transferred into flasks and 1.6 ml of combined C-sources and vitamins (2.3.1.1.14) was added under oxic conditions. The flasks were sealed with an autoclaced air-permeable cellulose stopper and had an additional protuberance that enables the non-invasive the measurement of the optical density (OD; 2.3.2.4) during growth.
2.3.1.2.5. DE/ISO/NO3-medium for growth experiments with isolates
Yeast 0.06 g
Glucose 0.09 g
Mineral salts DE-B (2.3.1.1.4) 1 ml
Trace elements DE (2.3.1.1.6) 1 ml
Mineral salts DE-A (2.3.1.1.3) ad 100 ml
The DE/ISO/NO3-medium was used for growth experiments under anoxic conditions with nitrate as electron acceptor (2.3.2.2.2). The anoxic medium (modified from Atlas & Parks [2000]) was prepared in serum vials flushed with 100 % N2 before, during, and after the application of the components to gain anoxic conditions. The pH was adjusted to 6.8 - 7.0, and the solution was autoclaved. After cooling down, 14 ml medium was transferred into sterile anoxic tubes that were flushed with sterile 100 % N2 before and during the procedure.
For each anoxic tube, 1.6 ml of combined C-sources, vitamins, and nitrate (2.3.1.1.15) was added.
2.3.1.2.6. RUP-medium for the enrichment and isolation of methanogens
Yeast 0.015 g
Tryptone 0.015 g
Mineral salts ME (2.3.1.1.5) 1.5 ml
Trace elements ME (2.3.1.1.7) 0.6 ml
Vitamins ME-A (2.3.1.1.9) 30 µl
Vitamins ME-B (2.3.1.1.10) 3 µl
Resazurin (0.1 %) 0.3 ml
Bicarbonate 4.5 g
Soil extract (2.3.1.1.16) 1.5 ml
Cysteine solution (7.5 %) 1.2 ml
Na2S solution (15 %) 0.6 ml
ddH2O ad 300 ml
For the RUP-medium (modified from Wüst et al. [2009c] and Bräuer et al. [2006]) used for the enrichment and isolation of methanogens (2.3.2.3), the medium was prepared in serum vials flushed with 100 % N2 before, during, and after the application. Boiling ddH2O was added to all components except for the soil extract, cysteine, Na2S, and bicarbonate
(NaHCO3). After cooling down with a permanent flow of N2, the soil extract, cysteine, Na2S, and bicarbonate were added. The pH was adjusted to 7.1 with NaOH and a saturated solution of bicarbonate, and the medium was flushed with CO2 (100 %). After another boiling and cooling down with a permanent flow of 100 % CO2, the medium was filled into anoxic tubes (9 ml per 24 ml-vial) that were flushed with 100 % CO2 before and during the procedure. For each anoxic tube, 8 ml of 100 % H2 was added (the remaining gas phase consisted in the anoxic tube of CO2) and the anoxic tubes were autoclaved. After cooling down, 0.1 ml of the earthworm extract (2.3.1.1.17) was added. The tubes were subsequently inoculated (2.3.2.3). For the very first enrichment step, i.e., the inoculum with material from E. eugeniae (2.3.2.3), a medium was used that contained no earthworm extract but the 20-fold amount of yeast and tryptone.
2.3.1.2.7. SOC medium
Tryptone 2 g
Yeast extract 0.5 g
NaCl solution (1 M) 1.0 ml
KCl solution (1 M) 0.25 ml
Mg2+ solution (2 M) 1.0 ml
Glucose solution (2 M) 1.0 ml
ddH2O ad 100 ml
Tryptone, yeast extract, NaCl and KCl were filled up with ddH2O to approximately 95 ml and autoclaved. Sterile filtered (0.2 µm pore diameter) Mg2+ and glucose solutions were added, the pH was adjusted to 7.0 with sterile filtered solutions, and sterile filtered ddH2O was added up to a final volume of 100 ml (Green & Sambrook 2012). Aliquots of the SOC medium were transferred into sterile 2 ml Eppendorf tubes and stored at - 20 °C.
2.3.1.2.8. LB (lysogeny broth) plates
Tryptone 10 g
Yeast extract 5 g
NaCl 5 g
Agar 15 g
ddH2O ad 1,000 ml
Tryptone, yeast extract, NaCl, and agar were filled up with ddH2O to approximately 980 ml. The pH was adjusted to 7.0 and ddH2O was added up to a final volume of 1,000 ml (Green & Sambrook 2012). After autoclaving, the medium was poured into sterile plastic Petri dishes and stored at 4 °C after solidification.
2.3.1.2.9. LB plates with ampicillin/IPTG/X-Gal
For blue/white screening of clones (2.5.9.3), 1 ml ampicillin (100 mg ml-1), 1 ml isopropyl-β-D-thiogalactopyranoside (IPTG; 0.5 M), and 1.6 ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 50 mg ml-1 in N,N´-dimethylformamide) solution was added to an autoclaved LB medium (2.3.1.2.8) after cooling down to approximately 60 °C. The medium was poured into sterile plastic Petri dishes, and the 'AIX plates' were stored at 4 °C after solidification.