Editing DSMZ Cells (section)

== <span style="line-height: 11.8857135772705px;">T. neapolitana</span> ==

The following medium (modified from ATCC 1977 medium recommended for T. elfeii) was used<br/>to prepare the batch reactors for culture of T. neapolitana: NH4Cl, 1.0 gm: K2HPO4 , 0.3 gm:<br/>KH2PO4 , 0.3 gm: MgCl2 x 6H2 O, 0.2 gm: CaCl2 x 2H2O, 0.1 gm: NaCl, 10.0 gm: KCl, 0.1 gm:&nbsp;Cysteine HCl, 1.0 gm: Yeast Extract, 2.0 gm: Trypticase, 2.0 gm: Vitamin Solution (DSMZ&nbsp;medium 141), 10.0 ml: Trace Element Solution (DSMZ medium 141), 10.0 ml: H 2 O, 1.0 L. The&nbsp;initial pH of the medium was adjusted to 8.5 @ 20°C with NaOH. Trizma base (1.0E-04M) can be&nbsp;added to help maintain pH in an optimal range for longer periods of time during incubation. A&nbsp;primary carbon source (0.25 g/50 ml cellulose, cellobiose, starches, or glucose) was also added.

<br/>The medium was always prepared aerobically. Fifty ml. of the above medium was aliquoted into<br/>serum bottles, allowing 110 ml. headspace for gas accumulation. Excess oxygen was removed by&nbsp;heating the batch reactors containing medium and substrate to 100°C while sparging its contents (2-10 min) with nitrogen gas. Sparging time was varied to achieve predetermined concentrations of&nbsp;oxygen of between 1 and 12% in the headspace and then the actual oxygen concentration in the&nbsp;bottle was tested using a gas chromatograph. (The effects of different oxygen concentrations are&nbsp;shown below). Serum bottles were then sealed, capped and sterilized.



=== Culture conditions ===

Thermotoga maritima DSM 3109, T. neapolitana DSM 4359, and T. elfii DSM 9442 were grown in&nbsp;a modified version of the ATCC 1977 medium (see van Ooteghem et al. 2002) containing 1 g<br/>cysteine.HCl l -1 as reducing agent, and glucose, xylose, arabinose or glycerol as carbon substrate.<br/>The medium also contained 2 g yeast extract l -1 and 2 g peptone l -1 , which are needed by these&nbsp;bacteria. Potassium phosphate was increased from 0.6 to 6.3 g l -1 to increase the buffering capacity&nbsp;of the growth medium (Eriksen et al. 2008), while the concentrations of MgCl2.H2 0 and CaCl2 .H20&nbsp;were reduced to 0.2 and 0.01 g l -1 , respectively, to prevent formation of precipitates. MgCl, CaCl2&nbsp;and the carbon source (glucose, xylose, arabinose, or glycerol) were autoclaved in a separate&nbsp;solution before mixed with a solution containing the remaining medium constituents. A few crystals&nbsp;of resazurin were added as redox indicator&nbsp;<br/>Batch cultures of Thermotoga maritima, T. neapolitana, and T. elfii were grown in 120 ml serum&nbsp;bottles containing 60 ml medium with shaking at 200 rpm and 80 o C. Before the cultures were&nbsp;inoculated, the bottles were sealed by butyl rubber stoppers and flushed with N 2 .<br/>Cultures of T. neapolitana were also grown in a 3 l Applikon bioreactor containing&nbsp;2.75 l medium. The reactor was equipped with a Pt100 temperature sensor and an autoclavable pH&nbsp;electrode (Mettler Toledo). Temperature and pH were automatically controlled by a heating &nbsp;blanket&nbsp;wrapped around the reactor wall, and by titration with 1 M NaOH. Cultures were stirred by a 4&nbsp;bladed Rushton turbine and aerated by headspace or by sparger aeration with nitrogen gas. The exit&nbsp;gas was passed through a condenser at 4 o C to reduce evaporation before it was passed through on-line sensors for quantification of gas-flow rate (AWM3000, Honeywell), and hydrogen (EZT3HYE&nbsp;'Easy Cal' CiTiceL) and CO 2 (IRcel, City Technology, Portsmouth, UK) in the off-gas.



Two hyperthermophilic bacteria, viz. Thermotoga maritima strain DSM 3109 and Thermotoga<br/>neapolitana strain DSM 4359, were analyzed for H 2 production.<br/>Anaerobic batch cultivations were performed in 120- and 240-mL serum bottles with a working<br/>volume of 25 mL or 50 mL, at constant temperature of 80oC and shaking at 200 rpm. Media were&nbsp;inoculated with a 10% (v/v) pre-culture. Growth on glucose (2.5 g/L) and glycerol (5 g/L) by T. maritima&nbsp;was tested on three different types of media, indicated as medium A1 [23], A2 [23], and M3. M3 media&nbsp;was a modified M2 [21] medium consisting of (amounts are in grams per liter of deionized water): 1.5 g&nbsp;KH 2 PO 4 ; 2.4 g Na 2 HPO 4 ·2H 2 O; 0.5 g NH 4 Cl; 0.2 g MgCl 2 ·6H 2 O; 2.0 mg NiCl 2 ·6H 2 O; 11.9 g HEPES (N-2-hydroxyethylpiperazine-Nƍ-2 ethanesulphonic acid); 2 g yeast extract (YE); 15 ml trace element&nbsp;solution (DSM-TES, see DSMZ medium 141, complemented with Na 2 WO 4 3.00 mg/L); 1.0 mL of&nbsp;vitamin solution (Biotin 2 mg, Nicotinamide 20 mg, p-Aminobenzoic acid 10 mg, Thiamine (Vit.B 1 ) 20&nbsp;mg, Pantothenic acid 10 mg, Pyridoxamine 50 mg, Cyanocobalamin and Riboflavin 10 mg [24]); 1.0 g/L&nbsp;of cysteine hydrochloride as reducing agent and 1 mg resazurin, which was used as a redox indicator.<br/>Anaerobic conditions were achieved by flushing the headspace of the serum bottles with pure N 2 for&nbsp;5 min. Growth on glucose (2.5 g/L) and glycerol (5g/L) by T. neapolitana was performed on M3 medium.<br/>The NaCl concentration used was 2.7% (w/v) for T. maritima and 2.0% (w/v) for T. neapolitana. The&nbsp;starting pH of the medium was adjusted to pH 6.9 for T. maritima and pH 7.3 for T. neapolitana with 10&nbsp;mM NaOH.<br/>The optimal growth parameters (pH, YE concentration) for glycerol (2.5 g/L) conversion by T.<br/>neapolitana was investigated on M3 medium for the pH range of 5-8.5 (YE, 2g/L) and YE concentration&nbsp;range 0- 4 g/L (pH7).