Editing DSMZ Cells (section)

=== Chinese Method 2 ===

''Thermotoga maritima'' (ATCC 43589) was tested for growth on: (i) modified Luria–Bertani (LB) medium containing 2.7% (w/v) NaCl, (ii) a common marine medium based on one-half-strength Turks Island salts (Merk Index no. 9954) with the modification of 2.7% (w/v) NaCl, 0.5% (w/v) soluble starch and 20 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) instead of 1,4-piperazine-''N, N''′-bis(2-ethanesulfonic acid) (PIPES) buffer, (iii) TBSY-HEPES medium, which is TB medium ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-6 Childers''et al.'',1992]) with the same modification as for the common marine medium, (iv) ''Thermoanaerobacter ethanolicus'' medium (ATCC 1190) supplemented with 2.7% NaCl (''Te''<sup>''+''</sup> medium), and (v) a simplified ''Te''<sup>''+''</sup> medium, in which the vitamin solution was omitted and Wolf's mineral solution was replaced with Balch's trace mineral solution ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-2 Balch''et al.'',1979]).

The ''T. maritima'' basal medium (TMB) contained, per liter, the following: 1.5 g KH<sub>2</sub>PO<sub>4</sub>; 4.2 g Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O (22 mM PO<sub>4</sub><sup>3−</sup>,); 0.5 g NH<sub>4</sub>Cl; 0.2 g MgCl<sub>2</sub>·6H<sub>2</sub>O (1 mM); 27 g NaCl; 4 g yeast extract (Oxoid Ltd, Basingstoke, UK); 1 mL Balch's trace mineral solution; 1 mg resazurin; 40 mL reducing solution. The reducing solution contained 200 mL NaOH (0.2 M); 2.5 g Na<sub>2</sub>S·9H<sub>2</sub>O; 2.5 g cysteine HCl·H<sub>2</sub>O. When required, thiosulfate was added to TMB from sterile stock solutions to a final concentration of 20 mM. The starting pH of the medium was 7.5 after adding the reducing solution. Anaerobic TMB was prepared in Hungate tubes or serum bottles, either inside the anaerobic chamber (Sheldon Manufacturing Inc., Cornelius, USA), or outside, using the Hungate technique ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-5 Braun''et al.'',1979]; [http://femsle.oxfordjournals.org/content/259/2/254.long#ref-8 Ljungdahl & Wiegel, 1986]). The carbohydrate, including soluble starch, xylan (from birch wood), carboxymethyl cellulose (CMC), glucose or xylose was added to the TMB to a final concentration of 0.5% (w/v) (TMB-starch, TMB-xylan, TMB-CMC, TMB-glucose, TMB-xylose). The cultures were incubated at 80°C after a 2% (v/v) typical inoculation. Samples were taken by sterile injector, and immediately cooled in an ice bath for further study.

In a 10-L fermentor, 7 L of non-reduced steam-sterilized TMB (121°C, 0.1 MPa, 40 min) was flushed with either filtrated nitrogen or carbon dioxide at 0.04 MPa, throughout cell growth. A 140 mL portion of the reducing solution was injected into the medium after its temperature had fallen to 75°C. Sterile soluble starch, glucose or xylose stock solution was added to the desired concentrations. Liquid fermentation was done with the temperature set to 75±1°C, the impeller at 100 r.p.m., and the pH to 6.5, with batch feeding of sterile 5 M NaOH. Samples were taken aseptically and immediately cooled in an ice bath for further study.

Xylan, CMC, HEPES and resazurin were purchased from Sigma (St Louis, MO, USA). All other chemicals of reagent grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).



The T. maritima basal medium (TMB) contained, per liter, the following:

1.5 g KH2PO4&nbsp;;

4.2 g Na2HPO4 . 12H2O (22 mM PO<sup>3</sup><sub>4</sub> ,);

0.5 g NH4Cl;

0.2 g MgCl2. 6H2O (1 mM);<br/>27 g NaCl;

4 g yeast extract<br/>1 mL Balch’s trace mineral solution;

1 mg resazurin;

40 mL reducing solution.

The reducing solution contained 200 mL NaOH (0.2 M); 2.5 g Na2S. 9H2O; 2.5 g cysteine HCl. H2O.<br/>When required, thiosulfate was added to TMB from sterile stock solutions to a final concentration of 20 mM. The starting pH of the medium was 7.5 after adding the reducing solution. Anaerobic TMB was prepared in Hungate tubes or serum bottles, either inside the anaerobic chamber


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