TITLE: NLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria
AUTHORS: Markus De Raad, Yifan V. Li, Jennifer V. Kuehl, Peter F. Andeer, Suzanne M. Kosina, Andrew Hendrickson, Nicholas R. Saichek, Amber N. Golini, La Zhen Han, Ying Wang, Benjamin P. Bowen, Adam M. Deutschbauer, Adam Arkin, Romy Chakraborty, Trent R. Northen

[DESCRIPTION]
This is the protocol for the preparation of NLDM medium as described in de Raad et al., 2022 - A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria. 

The Northen Lab Defined Medium (NLDM) is a defined medium and thus suited for exometabolomic experiments. It is  based on the metabolite composition of R2A and soil, metabolite usage by microbes, and biologically relevant elemental stoichiometry of these compounds. NLDM supports the growth of a wide range of phylogenetically diverse soil bacterial isolates and all of its metabolites are trackable through LC–MS/MS analysis. This demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities. We anticipate that the NLDM medium will enable the examination of microbial substrate utilization for a broad range of isolates both directly and through dilution and amendments. We speculate that this media may have additional value in supporting microbial isolations and additional types of microbial characterization.

Please cite NLDM as follows: de Raad M, Li YV, Kuehl JV, Andeer PF, Kosina SM, Hendrickson A, Saichek NR, Golini AN, Han LZ, Wang Y, Bowen BP, Deutschbauer AM, Arkin AP, Chakraborty R and Northen TR (2022) A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria. Front. Microbiol. 13:855331. DOI:10.3389/fmicb.2022.855331

[STEPS]
SECTION: NLDM Components
1. NLDM stock solutions
See Materials section on how to prepare stock solutions

      Medium   components    NLDM Solution1 - 'Sugars' -   10X    NLDM Solution 2 -   'Organic_Acids' - 10X     NLDM Solution3 - 'Amino_Acid'   - 10X    NLDM Solution 4 -   'Nucleobase/Nucleoside' - 50X    NLDM Solution 5 - 'Other_Cpd'   - 50X    NLDM Salt solution 1 - 'MgSO4'   - 50X    NLDM Salt solution 2 - 'NH4Cl'   - 50X    NLDM Salt solution 3 - 'CaCl2'   - 50X    NLDM Salt solution 4 -   'PO4'  - 50X     Vitamin solution - 100X    Trace Mineral solution - 100X  
 
SECTION: NLDM PREPARATION
2. For the preparation of 500 mL NLDM medium mix the following stock solutions:

           Volume (mL)    NLDM Solution   1 - 'Sugars' - 10X  50    NLDM Solution   2 - 'Organic_Acids' - 10X  50    NLDM Solution   3 - 'Amino_Acid' - 10X  50    NLDM Solution   4 - 'Nucleobase/Nucleoside' - 50X  10    NLDM Solution   5 - 'Other_Cpd' - 50X  10    NLDM Salt   solution 1 - 'MgSO4' - 50X  10    NLDM Salt   solution 2 - 'NH4Cl' - 50X  10    NLDM Salt   solution 3 - 'CaCl2' - 50X  10    NLDM Salt   solution 4 - 'PO4'  - 50X  10    Vitamin   solution - 100X  5    Trace Mineral   solution - 100X  5    H2O  280    TOTAL  500 mL  
 
SECTION: NLDM pH
3. Adjust the pH of the NLDM medium 7.0 - 7.2 if needed
SECTION: STERILIZATION
4. Filter sterilize the NLDM medium using a 0.22 um filter
SECTION: STORAGE
5. Store the NLDM medium at 4ºC in the dark