Jul 01, 2020

Public workspacePhenzine Oxidizer Enrichment and Isolation

DOI
dx.doi.org/10.17504/protocols.io.bh4tj8wn
  • 1California Institute of Technology;
  • 2Weizmann Institute of Science
Lev Tsypin
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DOI: dx.doi.org/10.17504/protocols.io.bh4tj8wn
Protocol Citation: Lev Tsypin, Yinon Bar-On, Scott Saunders, Jared R Leadbetter, Dianne K Newman 2020. Phenzine Oxidizer Enrichment and Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.bh4tj8wn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol was used during the 2017 Microbial Diversity course at the Marine Biological Laboratory in Woods Hole, MA.
Created: July 01, 2020
Last Modified: July 01, 2020
Protocol Integer ID: 38771
Abstract
This is a protocol for the enrichment of phenazine-1-carboxylic acid oxidizing microbes from a soil sample.
Medium compostion
Medium compostion
Stock solutions

100x Freshwater salts:
  • 1.71 M sodium chloride
  • 197 mM magnesium chloride
  • 68 mM calcium chloride
  • 671 mM potassium chloride

1000x Trace Elements:
  • 20 mM HCl
  • 7.5 mM FeCl3 6H2O
  • 480 uM H3BO3
  • 500 uM MnCl2 4H2O
  • 6.8 mM CoCl2 6H2O
  • 1 mM NiCl2 6H2O
  • 12 uM CuCl2 2H2O
  • 500 uM ZnCl2
  • 23 uM Na2SeO3
  • 150 uM Na2MoO4

1000x 13-vitamin solution
  • 1 mM MOPS pH 7.2
  • 100 µg/mL riboflavin
  • 30 µg/mL biotin
  • 100 µg/mL thiamine HCl
  • 100 µg/mL L-ascorbic acid
  • 100 µg/mL d-Ca-pantothenate
  • 100 µg/mL folic acid
  • 100 µg/mL nicotinic acid
  • 100 µg/mL 4-aminobenzoic acid
  • 100 µg/mL pyridoxine HCl
  • 100 µg/mL lipoic acid
  • 100 µg/mL Nicotinamide adenine dinucleotide
  • 100 µg/mL thiamine pyrophosphate
  • 10 µg/mL cyanocobalamin

2 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 color
  • Stir 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 solution
  • 1x freshwater salts solution
  • 1x 13-vitamin solution

Isolation medium (oxic or anoxic, agar plates)
  • 1 mM sodium phosphate buffer pH 7
  • 10 mM ammonium chloride
  • 100 uM sodium sulfate
  • 1 mM sodium acetate
  • 10 mM sodium nitrate
  • 1x freshwater salts solution
  • 1x trace elements solution
  • 1x 13-vitamin solution
  • 1.5 % agar

Sampling
Sampling
Collect topsoil by scooping directly with 50 mL conical tube.
Grind with mortar and pestle
Pass through 2 mm sieve to remove large rocks and organic matter.
Transfer to anaerobic chamber
Inoculation of enrichment cultures
Inoculation of enrichment cultures
Prepare 96-well plate with 190 uL of anoxic enrichment medium per well.
Add 20 mg soil into each well.
Prepare Balch tubes with 9 mL of anoxic enrichment medium in each.
Add 1 g soil into each tube and seal.
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.
Passage candidate PCA-oxidizing enrichments via 1:10 dilutions into fresh medium, maintaining anoxic conditions.
Isolation
Isolation
Plate enrichment cultures onto LB/Agar and the solid isolation medium.
Grow both oxically and anoxically in an anaerobic chamber.
Pick colonies of different morphologies and assay PCA-oxidizing capability in the enrichment medium as a pure culture.
Select isolates that oxidize PCA in pure culture for further study.