ANAEROBIC MEDIUM FOR DESULFITOBACTERIUM. STOCK SOLUTIONS Modified from DSMZ Medium 720

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1 Desulfitobacterium sp. is a strict anaerobic bacterium. For culturing this species (and also for another anaerobic microorganisms), you have to maintain Oxygen free conditions during cultivation. The main things that you have to keep in mind are: Make all the solutions in anaerobic water Put overpressure in the head space of your cultures, flushing with N 2 /CO 2 (replacing the oxygen) ANAEROBIC WATER Boil demi water for minutes Close with a loose lid, (e.g. aluminium foil) Cool to room temperature flushing with N 2 or 80%N 2 :20%CO 2 Make evacuated, sterile bottles (for culturing): To 21 x 120 ml bottle, add 1 ml demi water, cap, close, flux with 80%N 2 /20%CO 2 (overpressure 0.5atm) and autoclave (to avoid breaking bottles during autoclaving). STOCK SOLUTIONS Modified from DSMZ Medium 720 Store at 4 o C (except bicarbonate solution, PCE and Cl OHPA, store at room temperature) HCl (25%; 7.7 M) 10 ml FeCl 2 x 4 H 2 O 1.50 g ZnCl 2 70 mg MnCl 2 x 4 H2O 100 mg H 3 BO 3 6 mg CoCl 2 x 6 H 2 O 190 mg CuCl 2 x 2 H 2 O 2 mg NiCl 2 x 6 H 2 O 24 mg Na 2 MoO 4 x 2 H 2 O 36 mg EDTA, 0.5 g Distilled water 990 ml Adjust ph to 7.0 Trace element solution SL 10 (DSMZ 320) 1

2 First dissolve FeCl 2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000 ml No Need to autoclave 0.5 g NaOH 3 mg Na 2 SeO 3 x 5 H 2 O 4 mg Na 2 WO 4 x 2 H 2 O 1 l anaerobic water No need to autoclave Selenite/tungstate solution (DSMZ 385) NaHCO 3 : 1.55M (130 g) Bicarbonate solution Filter sterilisation into anaerobic bottle (for batch cultures), or autoclave (? Not yet tried). Keep at room temperature, can precipitate at lower temperature Only needed for continuous cultures, for batch experiments can be mixed directly into the medium Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine HCl 10.0 mg Thiamine HCl x 2 H 2 O 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg D Ca pantothenate 5.0 mg p Aminobenzoic acid 5.0 mg Lipoid acid 5.0 mg Add Distilled and anaerobic water 100 ml Filtersterilise into anaerobic bottle Vitamin solution 2

3 Vitamin B12 Cyanocobalamine (Vit. B12) 5.0 mg Distilled anaerobic water 100 ml Flush with anaerobic gas Autoclave Be aware that all bottles with vitamins must be in brown bottles or covered with aluminum foil. Na 2 S x 9 H 2 O (3%, w/v), 100 ml Filtersterilise into anaerobic bottle Use gloves and be careful!!! Work in fumehood. Oxygen scavenger: Sodium sulphide or L cysteine Use sulphide solution only with batch cultures, don t put into the chemostat!!! Sulphide can cause corrosion in the fermentor!! Reductant compound for continuous culture use L Cysteine: 0.4g/l final concentration 0.8 M L cysteine (9.7 g L Cysteine in 100 ml anaerobic water) Flush to remove traces of oxygen Autoclave Keep at room temperature, will otherwise precipitate. 500 mg/liter resazurin (7 Hydroxy 3H phenoxazin 3 one 10 oxide) is a blue dye (oxygen indicator) You can prepare 50ml and store it in a Falcon tube at 4 o C Resazurin solution 3

4 Yeast extract stock Prepare 4% stock solution of Yeast extract in anaerobic water and flush the bottle as the others. Filter sterilize and autoclave again, since yeast extract can contain spores. PCE stock (Desulfitobacterium hafniense Y51) Tetrachloroethylene / perchloroethylene (PCE) Mol weigth PCE= g/mol, density g/ml 1L pure PCE is mol = mol/ml Add 48 ml Hexadecane to a 120 ml bottle Close with a Viton stopper and Flush with N 2 Autoclave Add 2 ml pure PCE Final concentration of stock is 0.44M (0.5ml of this solution gives approximately 10mM PCE in the bottle, assuming approximately 20ml in the bottle). Take in account the head space if you are culturing this dehalogenating microorganism with PCE (you also can use another electron acceptor, such as Fumarate), important parameter if you want to monitor it s degradation by GC measurements. Cl OHPA (Desulfitobacterium hafniense DCB 2) 3 chloro 4 hydroxy phenylacetic acid (Cl OHPA) Mw= 186.6g Final concentration of stock 0.5M (don t concentrate further, can explode!) Add 9.33g Cl OHPA to 90 ml anaerobic demi water Dissolve Cl OHPA, can be hard to dissolve (stirring at ºC) Adjust ph to 7.5 with NaOH. The initial ph is around 3 Add anaerobic water up to 100 ml Close with Viton stopper. Shift gas phase in bottles to N2 (0.5 atm. overpressure) Autoclave Store in the dark at RT Chlorinated compounds are highly volatile; always work in the fume hood and use nitrile gloves. Use the special containers (dark blue) to put the waste. 4

5 BASAL MEDIUM PREPARATION (for batch experiments) Basal Medium modified from DSM medium for Desulfitobacterium hafniense (per liter) NH 4 Cl, 1.0 g K 2 HPO g MgSO 4 x 7H 2 O 0.1 g CaCl 2 x 2 H 2 O 0.05 g NaHCO 3, 2.60 g Resazurine stock 1ml Trace element solution SL 10 1ml Selenite/Tungstate solution 1ml L Lactic acid 1.70ml/l (20mM) (electron donor) Optionally, for alternative electron acceptors: NaNO g (10 mm) (as electron acceptor instead of chlorinated hydrocarbon like PCE) Optionally, for chloride poor culturing (for PCE experiments, to measure chloride production in addition to the chlorinated compounds), replace the above basal medium by (Gerritse et al): Ca(NO 3 ) 2, 0.05 g NH 4 H 2 PO g MgSO 4 x 7H 2 O 0.1 g Resazurine stock 1ml Trace element solution SL 10 1ml Selenite/Tungstate solution 1ml L Lactic acid 1.70ml/l (20mM) (electron donor) The complete consumption of C source (Lactate 20mM, converted to 20 mm acetate) requires to reduce 16 mm of Nitrate to nitrogen ( if we assumed that biomass production does not consume or produce electron/hydrogen, that it is not totally true). 10 mm of Nitrate will give electron acceptor limiting conditions Dissolve with anaerobic water and mix it gently. Cultures in 120ml serum bottles: 5

6 Dispense the medium (frequently we use 20ml or 50ml ) into 120 ml bottle. Swirl, gently to not mix O 2 into the medium Flush the medium in the serum bottles with 90%N 2 /10%CO 2 for 5 minutes (to remove possible oxygen during the transfer) Caps: butylrubber normally (grey and soft ones), but if the experiment is with PCE or Cl OHPA use the hard one (viton) as they are not permeable or sorb. Flush again with 90%N 2 /10%CO 2 ( overpressure 0.5 atm) Autoclave Complete the media: Working sterile: work near flame, connect filter (if it is needed) and needle next to it, use cotton with 97% of ethanol to flame the stopper. Let first cool down to room temperature after autoclaving the medium, this is important before adding bicarbonate and the vitamins. Complete the medium using syringes and continuous N 2 flushing to keep anaerobic conditions. Add vitamins (sterilized by filtration), sulfide (from sterile, anoxic stock solution) in case of batch cultures (use cysteine for chemostat, see above) and bicarbonate (from a sterile, anoxic stock solution prepared under 90%N2 and 10% CO2) (Per liter): Na 2 S x 9 H 2 O (3%, w/v) 10ml After adding the sulfide solution the medium becomes colorless after a while. If the liquid is pink it means that: there are traces of oxygen in the bottle or indicates wrong ph Vitamin solution: 1 ml Vitamin B 12 : 1 ml Yeast extract: 5ml (final concentration 0.02%) Electron acceptor: 10mM PCE (Desulfitobacterium hafniense Y51) 10mM Cl OHPA (Desulfitobacterium hafniense DCB 2) 6

7 Other electron acceptors/donors can be used Acceptors: Fumarate, Nitrate Donor: Pyruvate, Formate Inoculum: Add 5% of inoculum in your medium (1ml in ~20 ml of medium). Incubation: Incubate statically at 37 ºC usually it takes 2 5 days to get a fully grown culture depending the substrates. BASAL MEDIUM PREPARATION (for chemostats, and fermentor) Basal Medium modified from DSM medium for Desulfitobacterium hafniense (per liter) NH 4 Cl, 1.0 g K 2 HPO g MgSO 4 x 7H 2 O 0.1 g CaCl 2 x 2 H 2 O 0.05 g Resazurine stock 1ml Trace element solution SL 10 1ml Selenite/Tungstate solution 1ml L Lactic acid 1.70ml/l (20mM) (electron donor) Optionally: NaNO g (10 mm) (as electron acceptor instead of chlorinated hydrocarbon like PCE) Optionally, for chloride poor culturing conditions (for PCE experiments, to measure chloride production in addition to the chlorinated compounds), replace the above basal medium by (Gerritse et al): Ca(NO3) 2, 0.05 g NH 4 H 2 PO g MgSO 4 x 7H 2 O 0.1 g Resazurine stock 1ml Trace element solution SL 10 1ml Selenite/Tungstate solution 1ml L Lactic acid 1.70ml/l (20mM) (electron donor) 7

8 Autoclave Flush with anaerobic gas (90%N 2, 10%CO 2 ), overnight (~2 10 l/h) Working sterile: work near flame Complete the media: Complete the medium using syringes to take required volumes from anaerobic stock solutions, and continue N 2 /CO 2 flushing to keep anaerobic conditions. Add vitamins (sterilized by filtration), use cysteine (not sulfide) for chemostat, see above and bicarbonate (from a sterile, anoxic stock solution prepared under 90%N 2 and 10% CO 2 ) (Per liter): Bicarbonate solution 20ml (final concentration in the medium 2.6 g/l) L Cysteine (0.8 M) 4 ml Vitamin solution: 1 ml Vitamin B 12 : 1 ml Yeast extract: 5ml (final concentration 0.02%) Electron aceptor (Only to the fermentor, add as pure compound in case of PCE): 10mM PCE (Desulfitobacterium hafniense Y51) 10mM Cl OHPA (Desulfitobacterium hafniense DCB 2) Let s flush for a few hours more, prior to connecting to the fermentor (medium supply) or inoculation (fermentor) Inoculum for fermentor: Add 5% of inoculum into fermentor 8