Vertical-BLAST

These experiments were conducted in the laboratory of the research group of Prof:

Dr. Sylvia Schnell (General and Soil Microbiology) of the Institute of Applied Microbiology at the Justus Liebig University of Giessen

3.1.1. Origin of microorganisms

The methanogenic culture of Methanosarcina barkeri were brought from German Collection of Microorganisms and Cell Culture Braunschweig (DeutscheSammlung von Mikroorganismen und Zellkulturen DSMZ)

3.1.2 Used chemicals

Microelements Sl 10 solution contained (in mg per liter): FeCl2. 4H2O (1000); ZnCl2

(70); CoCl2 .6H2O (130), NaMoO4 2H2O (36); H3BO3 (6); MnCl2 4H2O (100) CuCl2 2H2O (2), NiCl2 .6H2O (24) and HCl 25% solution (10 ml) in 1000 ml deionised water. The solution was sterilized in the autoclave (La-VA-ncs 2003, Wolf Adolf Sanoclav, Bad Überkingen, Germany) for 25 min at 121°C (Widdel, et al., 1983).

Selenite-Tungstate solution (Widdel et al., 1983) contains (in mg per liter): NaOH (400); Na2SeO 5 H2O (6) and Na2WO4 2H2O (8). The solution was sterilized in the autoclavefor 25 min at 121°C.The seven mix-vitamins solution contained (in mg per liter): pyridoxamine-di-hydrochloride (200), lipoic acid (50) nicotinic acid (200); Ca-D (+)-pantothenic acid (100), 4-aminobenzic acid (80); D (+)-biotin (20) and cyanocobalamine (10). The solution was filter-sterilized and stored at 4°C in the dark (Widdel and Pfennig, 1981). Thiamine solution contained (in mg per liter): Thiamine dihydrochloride (100), Na2HPO4/H3PO4, with pH value 3.4 (25 mM). The solution was filter-sterilized with whaman^® filter (pore size, 0.2 μm, Whatman^®, Dassel, Freiburg, Germany) in sterile 50 ml bottles and stored at 4°C in the dark (Widdel and Bak, 1992). Riboflavin solution contained (in mg per liter): riboflavin (50) and acetic acid (20mM). The solution was filter-sterilized through Whatman^® (pore size 0.2 μm) filter (Whatman^®, Dassel, Freiburg, Germany) in sterile 50 ml bottles and stored at 4°C in the dark. The vitamin B12 solution contained (mg per liter) cyanocobalamine (50) and deionized water. The solution was filter-sterilized through nitrocellulose membrane (pore size, 0.2 μm) in sterile 50 ml bottles and stored at 4°C in the dark. For the

preparation of the bicarbonate solutions 84.0 g of NaHCO3 was dissolved in 1000 ml pure dionized water under CO2 atmosphere and portioned in a volume of 30 and 60 ml in screw cap serum bottles, leaving approximately 1/3 of the bottle volume as gas head space. The head space was flushed and exchanged to saturated the solution with CO2 by repeated flushing and vigorous shaking for 1-2 min. Lastly the solution portions were autoclaved and stored at room temperature (Pfennig 1978). Pure colorless crystals of Na2S.9H2O was flushed with deionized water using a plastic sieve, weighted in and dissolved in pure deionized water at a final concentration of 240.12 g/1000 ml the solution was portioned in small narrow vial each contains 5 ml then it flushed with mixture of nitrogen and carbon dioxide gas and closed tight before autoclaving. Sodium sulfide acts as a reducing agent. For yeast extract 2.5 g of yeast was weighted, dissolved in 50 ml of deionized water, mix thoroughly in glassed beaker, lastly put in serum bottle, closed and crimped with black butyl rupper stopper, and aluminum crimp respectively, autoclaved for 20 min under 121 °C. For casitone extract 2.5 g of casitone was weighted, dissolved in 50 ml of deionzied water, mix thoroughly in glassed beaker, lastly it was put in a serum bottle, closed with black butyl rubber stopper, and crimped with aluminum crimp, autoclaved for 20 min under 121°C. Pure methanol (99, 9%) was sterilized by using Whatman^® filter spore size 0.2 μm (Whatman^®), Dassel, Freiburg, Germany) put in a 120 ml serum bottle, stoppered with black butyl rubber and crimped with aluminum crimp (All vitamins and other solutions were provided either by Dr. Ratering or Mr. Schneider from the work group of Prof. Dr. Schnell).

3.1.3 Preparation of the oregano extract

The oregano leaves were dried under the shade and ground to fine powder to pass a 1mm sieve by using a laboratory electric mill. A sample was extracted with 99%

methanol and filtered after 24 h under shaking (Infors AG CH – 4103 BOTT MINEN);

the speed of the shaker was 150 rpm. The plant residue was re-extracted with the addition of 99% methanol for 24 h, and filtered again. Filtrates were combined together and concentrated on a rotary evaporator (BÜCHI Rotovap^®) at 42ºC to eliminate the methanol. Later the extracts were saved in sterile bottles under the cooled conditions until the use. The extracts' dry weight were achieved by evaporation the methanol and the concentration in mg/ml was determined according to Betoni et al. (2006).

3.1.4 Preparation of anaerobic liquid medium

Methanosarcina barkeri cultures were cultivated in anoxic, bicarbonate-buffered, sulfide-reduced, sterilized mineral medium (Widdel and Bak, 1992) containing:

1.0 g/l NaCl , 0.4 g/l MgCl2 6H2O,0.15 g/l CaCl2 2H2O 0.5 g/l KCl, 0.2 g/l KH2PO4; and 0.25 g/l NH4Cl. The medium was reduced with Na2S9H2O. The medium is prepared in a Widdel flask (1 or 2 liter volume) and autoclaved for 46 min at 121°C.

Then after autoclaving the medium was cooled down to the room temperature under nitrogen atmosphere by flashing the headspace with oxygen-free gas mixture of N2/CO2 (80/20, v/v, from Air Liquide, Duesseldorf, Germany). Addition of oxygen free gas is to remove the oxygen from the headspace. After cooling under the room temperature and still connecting to the gas line a NaHCO3 solutions (30ml/l), (1ml/l) of each of vitamin B12, Seven-vitamin mix solution, riboflavin , thiamin, trace elements solution SL10, selenite and tungstate solution, and Na2S 9 H2O (5 ml/l) were added to the medium. The pH value (Microprocessor pH meter-pH 535 MultiCal^®, WTW, Weilheim, Germany) of the medium was adjusted to 6.8 with the addition of 1 M HCl (Widdel et al., 1983).

For the cultivation of the methanogenic microorganisms, the medium was filled to 50 ml into each of 160 ml sized serum bottle (Ochs, Bovenden, Germany) and then sealed with the butyl rubber septa (Sigma-Aldrich, Steinheim, Germany), and crimped with the aluminum crimps. The 50 –ml filled serum bottles were left at the room temperature on the laboratory bench in the dark for overnight before inoculating the methanogenic organisms. The next day 0.1 ml of methanol, 4.0 ml of casitone and 4.0 ml of yeast extract were added aseptically before inoculation of the methanogens. The media were inoculated with 5.0 ml of a freshly grown stationary- phase culture. Kanamycin (0.5 mg/ml) per-culture (serum bottle) was added by N2/CO2 (80:20 [vol: vol]) gassed sterile syringes to make sure that the culture stay pure, and no other microbes invading the culture. The culture serum bottles were incubated statically and vertically but shaken manually from time to time briefly.

Cultures were routinely controlled by the optical density measurements and the microscopic examination (Axioscope; Zeiss, Jana, Germany). Optical densities of the cultures were measured after retrieving of one ml by N2-flushed syringe in plastic cuvettes (Brand, Wertheim).

Fig 6 Flask for preparation of anaerobic media for cultivation of methanogens (Widdel 1980)

3.1.5 Addition of the tested materials

The investigated materials were representative samples (well homogenized) and they were added aseptically by sterile N2/CO2 mix gas flushed syringe through the black rubber stopper after the culture well grown (after 4 to 6 days) and the reason behind that is to make sure that all the cultures used in the experiment are active.

Incubation occurred in 36°C in the dark and cultures were routinely checked and shacked manually from time to time.

3.1.6 Gas chromatography (GC) analysis

The concentration of methane produced by Methanosarcina barkeri was measured with the gas chromatograph (GC) Autosystem XL, Arnel (Perkin Elmer, Überlingen, Germany) combine with a flame-ionization detector (FID). The sample was taken directly from the head space of the serum bottle, after inserting the needle through the septum of the stopper. The sample was taken in a tight Pressure-Lok^® series A-2 (Supelco, Oakville, Canada) gas sampling syringes (with side–opened needle to prevent coring of gas chromatography septa and it had zero dead volume). Samples were injected manually in the injector by injection of 20 μl. Nitrogen (30 ml min^-1) was used as a carrier gas on a packed (80/100 mesh) carboxen (5 Ă, Serva, Heidelberg, Germany) with hydrogen (30 ml min^-1) and air mix flow (300 ml min^-1) as burning gases. The column (3 m x 4 mm) heated to 50°C where as the temperature of injector and detector was 230°C.

Gas production in the head spaces of the serum bottles was measured every three days using the syringe technique. The gas sample was identified and quantified by

comparing the chromatograms (from the GC run) with the chromatogram of an external methane standard (100.4 ppmv, from Duste-Steininger, Muelhausen, Germany) by the retention time and the peak area respectively. The certified standard sample of methane is used to compare and to calculate the concentrations of methane that produced in the head spaces of the serum bottles by the methanogens. At each sampling time, triplicate samples from each culture bottle was analyzed, and the means were calculated.

3.2 Laboratory digestion