TITLE: Phenzine Oxidizer Enrichment and Isolation
AUTHORS: Lev Tsypin, Yinon Bar-On, Scott Saunders, Jared R Leadbetter,  Dianne K Newman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for the enrichment of phenazine-1-carboxylic acid oxidizing microbes from a soil sample.</div></div>

[STEPS]
?. [Medium compostion]
Stock solutions100x Freshwater salts:1.71 M sodium chloride197 mM magnesium chloride68 mM calcium chloride671 mM potassium chloride1000x Trace Elements:20 mM HCl7.5 mM FeCl3 6H2O480 uM H3BO3500 uM MnCl2 4H2O6.8 mM CoCl2 6H2O1 mM NiCl2 6H2O12 uM CuCl2 2H2O500 uM ZnCl223 uM Na2SeO3150 uM Na2MoO41000x 13-vitamin solution1 mM MOPS pH 7.2 100 µg/mL riboflavin30 µg/mL biotin100 µg/mL thiamine HCl100 µg/mL L-ascorbic acid100 µg/mL d-Ca-pantothenate100 µg/mL folic acid100 µg/mL nicotinic acid100 µg/mL 4-aminobenzoic acid100 µg/mL pyridoxine HCl100 µg/mL lipoic acid100 µg/mL Nicotinamide adenine dinucleotide100 µg/mL thiamine pyrophosphate10 µg/mL cyanocobalamin2 mM reduced phenazine-1-carboxylic acid (PCA)In a sealed Balch tube or serum vial, sparge 2 mM oxidized PCA with 80:20 (vol:vol) H2/CO2 over Pd (II) until yellow-green in colorStir with a magnetic stirbar or otherwise agitate during sparging and overnight afterwards.Enrichment medium (anoxic)45 mM sodium bicarbonate buffer (buffer)10 mM ammonium chloride (nitrogen source)1 mM potassium phosphate (phosphorus source)1 mM reduced phenazine-1-carboxylic acid (PCA, electron donor)100 uM sodium acetate (carbon source)50 uM sodium sulfide (sulfur source)1x trace elements solution1x freshwater salts solution1x 13-vitamin solutionIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agar1000x Trace Elements:20 mM HCl7.5 mM FeCl3 6H2O480 uM H3BO3500 uM MnCl2 4H2O6.8 mM CoCl2 6H2O1 mM NiCl2 6H2O12 uM CuCl2 2H2O500 uM ZnCl223 uM Na2SeO3150 uM Na2MoO41000x 13-vitamin solution1 mM MOPS pH 7.2 100 µg/mL riboflavin30 µg/mL biotin100 µg/mL thiamine HCl100 µg/mL L-ascorbic acid100 µg/mL d-Ca-pantothenate100 µg/mL folic acid100 µg/mL nicotinic acid100 µg/mL 4-aminobenzoic acid100 µg/mL pyridoxine HCl100 µg/mL lipoic acid100 µg/mL Nicotinamide adenine dinucleotide100 µg/mL thiamine pyrophosphate10 µg/mL cyanocobalamin2 mM reduced phenazine-1-carboxylic acid (PCA)In a sealed Balch tube or serum vial, sparge 2 mM oxidized PCA with 80:20 (vol:vol) H2/CO2 over Pd (II) until yellow-green in colorStir with a magnetic stirbar or otherwise agitate during sparging and overnight afterwards.Enrichment medium (anoxic)45 mM sodium bicarbonate buffer (buffer)10 mM ammonium chloride (nitrogen source)1 mM potassium phosphate (phosphorus source)1 mM reduced phenazine-1-carboxylic acid (PCA, electron donor)100 uM sodium acetate (carbon source)50 uM sodium sulfide (sulfur source)1x trace elements solution1x freshwater salts solution1x 13-vitamin solutionIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agar1000x 13-vitamin solution1 mM MOPS pH 7.2 100 µg/mL riboflavin30 µg/mL biotin100 µg/mL thiamine HCl100 µg/mL L-ascorbic acid100 µg/mL d-Ca-pantothenate100 µg/mL folic acid100 µg/mL nicotinic acid100 µg/mL 4-aminobenzoic acid100 µg/mL pyridoxine HCl100 µg/mL lipoic acid100 µg/mL Nicotinamide adenine dinucleotide100 µg/mL thiamine pyrophosphate10 µg/mL cyanocobalamin2 mM reduced phenazine-1-carboxylic acid (PCA)In a sealed Balch tube or serum vial, sparge 2 mM oxidized PCA with 80:20 (vol:vol) H2/CO2 over Pd (II) until yellow-green in colorStir with a magnetic stirbar or otherwise agitate during sparging and overnight afterwards.Enrichment medium (anoxic)45 mM sodium bicarbonate buffer (buffer)10 mM ammonium chloride (nitrogen source)1 mM potassium phosphate (phosphorus source)1 mM reduced phenazine-1-carboxylic acid (PCA, electron donor)100 uM sodium acetate (carbon source)50 uM sodium sulfide (sulfur source)1x trace elements solution1x freshwater salts solution1x 13-vitamin solutionIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agar2 mM reduced phenazine-1-carboxylic acid (PCA)In a sealed Balch tube or serum vial, sparge 2 mM oxidized PCA with 80:20 (vol:vol) H2/CO2 over Pd (II) until yellow-green in colorStir with a magnetic stirbar or otherwise agitate during sparging and overnight afterwards.Enrichment medium (anoxic)45 mM sodium bicarbonate buffer (buffer)10 mM ammonium chloride (nitrogen source)1 mM potassium phosphate (phosphorus source)1 mM reduced phenazine-1-carboxylic acid (PCA, electron donor)100 uM sodium acetate (carbon source)50 uM sodium sulfide (sulfur source)1x trace elements solution1x freshwater salts solution1x 13-vitamin solutionIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agarEnrichment medium (anoxic)45 mM sodium bicarbonate buffer (buffer)10 mM ammonium chloride (nitrogen source)1 mM potassium phosphate (phosphorus source)1 mM reduced phenazine-1-carboxylic acid (PCA, electron donor)100 uM sodium acetate (carbon source)50 uM sodium sulfide (sulfur source)1x trace elements solution1x freshwater salts solution1x 13-vitamin solutionIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agarIsolation medium (oxic or anoxic, agar plates)1 mM sodium phosphate buffer pH 710 mM ammonium chloride100 uM sodium sulfate1 mM sodium acetate10 mM sodium nitrate1x freshwater salts solution1x trace elements solution1x 13-vitamin solution1.5 % agar
?. [Sampling]
Collect topsoil by scooping directly with 50 mL conical tube.
?. [Sampling]
Grind with mortar and pestle
?. [Sampling]
Pass through 2 mm sieve to remove large rocks and organic matter.
?. [Sampling]
Transfer to anaerobic chamber
?. [Inoculation of enrichment cultures]
Prepare 96-well plate with 190 uL of anoxic enrichment medium per well.
?. [Inoculation of enrichment cultures]
Add 20 mg soil into each well.
?. [Inoculation of enrichment cultures]
Prepare Balch tubes with 9 mL of anoxic enrichment medium in each.
?. [Inoculation of enrichment cultures]
Add 1 g soil into each tube and seal.
?. [Inoculation of enrichment cultures]
For both the 96-well plates and Balch tubes, watch for a color change over the next days. The yellow-green reduced PCA will turn clear if oxidized.
?. [Inoculation of enrichment cultures]
Passage candidate PCA-oxidizing enrichments via 1:10 dilutions into fresh medium, maintaining anoxic conditions.
?. [Isolation]
Plate enrichment cultures onto LB/Agar and the solid isolation medium.
?. [Isolation]
Grow both oxically and anoxically in an anaerobic chamber.
?. [Isolation]
Pick colonies of different morphologies and assay PCA-oxidizing capability in the enrichment medium as a pure culture.
?. [Isolation]
Select isolates that oxidize PCA in pure culture for further study.