NLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria

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

Aug 12, 2022

NLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria

DOI

dx.doi.org/10.17504/protocols.io.14egn7o2yv5d/v1

Markus De Raad¹,
Yifan V. Li²,
Jennifer V. Kuehl¹,
Peter F. Andeer¹,
Suzanne M. Kosina¹,
Andrew Hendrickson¹,
Nicholas R. Saichek¹,
Amber N. Golini¹,
La Zhen Han¹,
Ying Wang¹,
Benjamin P. Bowen¹,
Adam M. Deutschbauer¹,
Adam Arkin^1,3,
Romy Chakraborty²,
Trent R. Northen^1,4

¹Lawrence Berkeley National Laboratory, Environmental Genomics and Systems Biology Division, Berkeley, CA, United States;
²Earth and Environmental Sciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA, United States;
³Department of Bioengineering, University of California, Berkeley, Berkeley, CA, United States;
⁴Lawrence Berkeley National Laboratory, Joint Genome Institute, Berkeley, CA, United States

Markus De Raad

Lawrence Berkeley National Laboratory

DOI: dx.doi.org/10.17504/protocols.io.14egn7o2yv5d/v1

External link: https://www.frontiersin.org/articles/10.3389/fmicb.2022.855331/full

Protocol Citation: 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 2022. NLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7o2yv5d/v1

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

We use this protocol and it's working

Created: July 01, 2022

Last Modified: August 12, 2022

Protocol Integer ID: 65799

Keywords: Defined medium, defined media, exometabolomics, liquid chromatography mass spectrometry, soil bacteria, R2A

Funders Acknowledgement:

Under contract number DE-AC02-05CH11231, this material by ENIGMA – Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research and used resources of the National Energy Research Scientific Computing Center, a Department of Energy Office of Science User Facility

Grant ID: DE-AC02-05CH11231

Abstract

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

Materials

GENERAL NOTES
Store NLDM solutions 1-5 at -80ºC
Store NLDM Salt solutions 1-4 at 4ºC
Both the Vitamin Solution and Trace Mineral Solution are from ATCC (ATCC® MD-VS™ and ATCC® MD-TMS™)

NLDM Solution 1-5
ABCDEFGHIJK
NLDM Solution 1 - 'Sugars' - 10X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg for 50 mL stock solution
1sucroseC12H22O11sugarSigmaS3929-1KG342.30.875108.75149.8
2D-glucoseC6H12O6sugarSigmaG8270-1KG180.160.875108.7578.8
3trehalose dihydrateC12H22O11 x 2H2OsugarSanta Cruz Biotechnologiessc-202368378.330.875108.75165.5
4myo-inositolC6H12O6sugarSigmaI5125-50G180.160.875108.7578.8
5xyloseC5H10O5sugarSpectrumXY110150.130.875108.7565.7
6D-mannitolC6H14O6sugarSigmaM4125-500G182.170.875108.7579.7
7n-acetyl glucosamineC8H15NO6sugarSigmaA4106-25G221.210.875108.7596.8
NLDM Solution 2 - 'Organic_Acids' - 10X 
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg for 50 mL stock solution
8sodium-DL-lactateC3H5NaO2organic acidSigma71720-5G112.060.525105.2529.4
9DL-Malic acidC4H6O5organic acidSigma240176-50G134.090.525105.2535.2
10Citric acidC6H8O7organic acidSigmaC4540-100G192.120.525105.2550.4
11Succinic acidC4H6O4organic acidSigmaS3674-100G118.090.525105.2531.0
12sodium pyruvateC3H3O3Naorganic acidSigmaP2256-25G110.040.525105.2528.9
13alpha ketoglutaric acidC5H6O5organic acidSigmaK1750-100G146.10.525105.2538.4
14L-CitrullineC6H13N3O3organic acidSigmaC7629-5G175.190.525105.2546.0
NLDM Solution 3 - 'Amino_Acids' - 10X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg for 50 mL stock solution
15L-AlanineC3H7NO2amino acidSigmaA7627-100G89.090.175101.757.8
16L-ArginineC6H14N4O2amino acidSigmaA8094-100G174.20.175101.7515.2
17L-AsparagineC4H8N2O3amino acidSigmaA0884-25G132.120.175101.7511.6
18L-Aspartic acidC4H7NO4amino acidSigmaA9256-100G133.10.175101.7511.6
19L-CysteineC3H7NO2Samino acidSigma30089-25G121.160.175101.7510.6
20L-Glutamic acidC5H9NO4amino acidSigma49449-100G147.130.175101.7512.9
21L-GlutamineC5H10N2O3amino acidSigmaG3126-100G146.140.175101.7512.8
22GlycineC2H5NO2amino acidSigma410225-50G75.070.175101.756.6
23L-HistidineC6H9N3O2amino acidSigmaH8000-10G155.150.175101.7513.6
24L-IsoleucineC6H13NO2amino acidSigmaI2752-25G131.170.175101.7511.5
25L-LeucineC6H13NO2amino acidSigmaL8912-25G131.170.175101.7511.5
26L-LysineC6H14N2O2amino acidSigmaL5501-25G146.190.175101.7512.8
27L- MethionineC5H11NO2Samino acidSigmaM5308-25G149.210.175101.7513.1
28L-PhenylalanineC9H11NO2amino acidSigmaP5482-25G165.190.175101.7514.5
29L-ProlineC5H9NO2amino acidSigmaP5607-25G115.130.175101.7510.1
30L-SerineC3H7NO3amino acidSigmaS4311-25G105.090.175101.759.2
31L-ThreonineC4H9NO3amino acidSigmaT8441-25G119.120.175101.7510.4
32L-TryptophanC11H12N2O2amino acidSigmaT8941-25G204.230.175101.7517.9
33L-TyrosineC9H11NO3amino acidSigmaT8566-25G181.190.175101.7515.9
34L-ValineC5H11NO2amino acidSigma94619-25G117.150.175101.7510.3
NOTE: Dissolve all amino acids in 4 mM HCl. Sonicate for 30 minutes to dissolve all amino acids
NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg for 50 mL stock solution
355-methyluridineC10H14N2O6nucleosideSigma535893-25G258.230.0175500.87511.3
36AdenineC5H5N5nucleobaseSigmaA8626-5G135.130.0175500.8755.9
37AdenosineC10H13N5O4nucleosideSigmaA4036-5G267.240.0175500.87511.7
38CytidineC9H13N3O5nucleosideSigmaC4654-5G243.220.0175500.87510.6
39CytosineC4H5N3OnucleobaseSigmaC3506-1G111.10.0175500.8754.9
40GuanineC5H5N5OnucleobaseSigmaG11950-100G151.130.0175500.8756.6
41GuanosineC10H13N5O5nucleosideSigmaG6264-5G283.240.0175500.87512.4
42HypoxanthineC5H4N4OnucleobaseSigmaH9636-5G136.110.0175500.8756.0
43InosineC10H12N4O5nucleosideSigmaI4125-100G268.230.0175500.87511.7
44ThymineC5H6N2O2nucleobaseSigmaT0895-5G126.110.0175500.8755.5
45UracilC4H4N2O2nucleobaseSigmaU1128-25G112.090.0175500.8754.9
46UridineC9H12N2O6nucleosideSigmaU3003-5G244.20.0175500.87510.7
47XanthineC5H4N4O2nucleobaseSigmaX0626-5G152.110.0175500.8756.7
48Xanthosine dihydrateC10H12N4O6nucleobaseSigmaX0750-5G320.260.0175500.87514.0
NOTE:  Dissolve all nucleobases and nucelosides in 0.08M NaOH 
NLDM Solution 5 - 'Other_Cpds' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg for 50 mL stock solution
49gamma-Aminobutyric acidC4H9NO2otherSigmaA2129-25G103.120.0175500.8754.5
50EctoineC6H10N2O2otherSigma81619-1G-F142.160.0175500.8756.2
51BetaineC5H11NO2amino acid derivSigma61962-50G117.150.0175500.8755.1
52N-acetyl-L-Glutamic acidC7H11NO5amino acid derivSigma855642-25G189.170.0175500.8758.3
53NicotinamideC6H6N2OvitaminSigma72340-100G122.120.0175500.8755.3
54Ornithine HClC5H12N2O2amino acid derivSigmaO2375-5G168.620.0175500.8757.4
55Shikimic acidC7H10O5otherSigmaS5375-1G174.150.0175500.8757.6
56spermidineC7H19N3otherSigmaS0266-1G145.250.0175500.8756.4
57taurineC2H7NO3SotherSigmaT0625-10G125.150.0175500.8755.5
58Trigonelline HClC7H7NO2 x HClotherSigmaT5509-5G173.60.0175500.8757.6
59L-Carnitine HClC7H16NO3 x HClotherSigmaC0283-1G197.660.0175500.8758.6
60Choline ClC5H14NOotherSigmaC7017-5G139.620.0175500.8756.1
61N-acetyl-L-GlutamineC7H12N2O4amino acid derivSigmaA9125-25G188.180.0175500.8758.2
62n-acetyl-L-lysineC8H16N2O3amino acid derivSigmaA4021-1G188.220.0175500.8758.2
63n-acetylmuramic acidC11H19NO8otherSigmaA3007-500MG293.270.0175500.87512.8
64sn-glycero-3-phosphocholineC8H20NO6PotherCayman20736257.220.0175500.87511.3

NLDM Salt Solution 1-4
ABCDEFGHIJK
NLDM Salt solution 1 - 'MgSO4' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg/50ml
S1Magnesium sulfate heptahydrateMgSO4 * 7H2OSaltSigmaM2773-500G246.470.8125040.6500.3
NLDM Salt solution 2 - 'NH4Cl' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg/50ml
S2Ammonium chlorideNH4ClSaltSigmaA9434-500G53.4915050133.7
NLDM Salt solution 3 - 'CaCl2' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg/50ml
S3Calcium chloride dihydrateCaCl2 * 2 H2OSaltSigmaC7902-500G147.010.685034249.9
NLDM Salt solution 4 - 'PO4' - 50X
#CompoundformulaclassSupplierSupplier #MW (avg)Final_Conc_mmol/LConc_FactorConc mmol/Lmg/50ml
S4Sodium phosphate monobasicNaH2PO4SaltSigmaS0751-500G119.981.6655083.25499.4
S5Potassium phosphate dibasicK2HPO4SaltSigmaP3786-500G174.183.3350166.51450.0

Vitamin Solution - 100X
Vitamin Supplement (ATCC® MD-VS™) based on Wolfe's Vitamin Solution
Order at ATCC 

Trace Mineral Solution - 100X
Trace Mineral Supplement (ATCC® MD-TMS™) based on Wolfe's mineral solution
Order at ATCC

NLDM Components

NLDM stock solutions
See Materials section on how to prepare stock solutions

A
Medium components
NLDM Solution 1 - 'Sugars' - 10X
NLDM Solution 2 - 'Organic_Acids' - 10X
NLDM Solution 3 - 'Amino_Acids' - 10X
NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X
NLDM Solution 5 - 'Other_Cpds' - 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
 

NLDM PREPARATION

For the preparation of 500 mL NLDM medium mix the following stock solutions:

AB
 Volume (mL)
NLDM Solution 1 - 'Sugars' - 10X50
NLDM Solution 2 - 'Organic_Acids' - 10X50
NLDM Solution 3 - 'Amino_Acids' - 10X50
NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X10
NLDM Solution 5 - 'Other_Cpds' - 50X10
NLDM Salt solution 1 - 'MgSO4' - 50X10
NLDM Salt solution 2 - 'NH4Cl' - 50X10
NLDM Salt solution 3 - 'CaCl2' - 50X10
NLDM Salt solution 4 - 'PO4'  - 50X10
Vitamin solution - 100X5
Trace Mineral solution - 100X5
Milli-Q Water280
TOTAL500
 

NLDM pH

Adjust the pH of the NLDM medium 7.0 - 7.2 if needed

STERILIZATION

Filter sterilize the NLDM medium using a 0.22 um filter

STORAGE

Store the NLDM medium at 4ºC in the dark

A	B	C	D	E	F	G	H	I	J	K
NLDM Solution 1 - 'Sugars' - 10X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg for 50 mL stock solution
1	sucrose	C12H22O11	sugar	Sigma	S3929-1KG	342.3	0.875	10	8.75	149.8
2	D-glucose	C6H12O6	sugar	Sigma	G8270-1KG	180.16	0.875	10	8.75	78.8
3	trehalose dihydrate	C12H22O11 x 2H2O	sugar	Santa Cruz Biotechnologies	sc-202368	378.33	0.875	10	8.75	165.5
4	myo-inositol	C6H12O6	sugar	Sigma	I5125-50G	180.16	0.875	10	8.75	78.8
5	xylose	C5H10O5	sugar	Spectrum	XY110	150.13	0.875	10	8.75	65.7
6	D-mannitol	C6H14O6	sugar	Sigma	M4125-500G	182.17	0.875	10	8.75	79.7
7	n-acetyl glucosamine	C8H15NO6	sugar	Sigma	A4106-25G	221.21	0.875	10	8.75	96.8

NLDM Solution 2 - 'Organic_Acids' - 10X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg for 50 mL stock solution
8	sodium-DL-lactate	C3H5NaO2	organic acid	Sigma	71720-5G	112.06	0.525	10	5.25	29.4
9	DL-Malic acid	C4H6O5	organic acid	Sigma	240176-50G	134.09	0.525	10	5.25	35.2
10	Citric acid	C6H8O7	organic acid	Sigma	C4540-100G	192.12	0.525	10	5.25	50.4
11	Succinic acid	C4H6O4	organic acid	Sigma	S3674-100G	118.09	0.525	10	5.25	31.0
12	sodium pyruvate	C3H3O3Na	organic acid	Sigma	P2256-25G	110.04	0.525	10	5.25	28.9
13	alpha ketoglutaric acid	C5H6O5	organic acid	Sigma	K1750-100G	146.1	0.525	10	5.25	38.4
14	L-Citrulline	C6H13N3O3	organic acid	Sigma	C7629-5G	175.19	0.525	10	5.25	46.0

NLDM Solution 3 - 'Amino_Acids' - 10X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg for 50 mL stock solution
15	L-Alanine	C3H7NO2	amino acid	Sigma	A7627-100G	89.09	0.175	10	1.75	7.8
16	L-Arginine	C6H14N4O2	amino acid	Sigma	A8094-100G	174.2	0.175	10	1.75	15.2
17	L-Asparagine	C4H8N2O3	amino acid	Sigma	A0884-25G	132.12	0.175	10	1.75	11.6
18	L-Aspartic acid	C4H7NO4	amino acid	Sigma	A9256-100G	133.1	0.175	10	1.75	11.6
19	L-Cysteine	C3H7NO2S	amino acid	Sigma	30089-25G	121.16	0.175	10	1.75	10.6
20	L-Glutamic acid	C5H9NO4	amino acid	Sigma	49449-100G	147.13	0.175	10	1.75	12.9
21	L-Glutamine	C5H10N2O3	amino acid	Sigma	G3126-100G	146.14	0.175	10	1.75	12.8
22	Glycine	C2H5NO2	amino acid	Sigma	410225-50G	75.07	0.175	10	1.75	6.6
23	L-Histidine	C6H9N3O2	amino acid	Sigma	H8000-10G	155.15	0.175	10	1.75	13.6
24	L-Isoleucine	C6H13NO2	amino acid	Sigma	I2752-25G	131.17	0.175	10	1.75	11.5
25	L-Leucine	C6H13NO2	amino acid	Sigma	L8912-25G	131.17	0.175	10	1.75	11.5
26	L-Lysine	C6H14N2O2	amino acid	Sigma	L5501-25G	146.19	0.175	10	1.75	12.8
27	L- Methionine	C5H11NO2S	amino acid	Sigma	M5308-25G	149.21	0.175	10	1.75	13.1
28	L-Phenylalanine	C9H11NO2	amino acid	Sigma	P5482-25G	165.19	0.175	10	1.75	14.5
29	L-Proline	C5H9NO2	amino acid	Sigma	P5607-25G	115.13	0.175	10	1.75	10.1
30	L-Serine	C3H7NO3	amino acid	Sigma	S4311-25G	105.09	0.175	10	1.75	9.2
31	L-Threonine	C4H9NO3	amino acid	Sigma	T8441-25G	119.12	0.175	10	1.75	10.4
32	L-Tryptophan	C11H12N2O2	amino acid	Sigma	T8941-25G	204.23	0.175	10	1.75	17.9
33	L-Tyrosine	C9H11NO3	amino acid	Sigma	T8566-25G	181.19	0.175	10	1.75	15.9
34	L-Valine	C5H11NO2	amino acid	Sigma	94619-25G	117.15	0.175	10	1.75	10.3
NOTE: Dissolve all amino acids in 4 mM HCl. Sonicate for 30 minutes to dissolve all amino acids

NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg for 50 mL stock solution
35	5-methyluridine	C10H14N2O6	nucleoside	Sigma	535893-25G	258.23	0.0175	50	0.875	11.3
36	Adenine	C5H5N5	nucleobase	Sigma	A8626-5G	135.13	0.0175	50	0.875	5.9
37	Adenosine	C10H13N5O4	nucleoside	Sigma	A4036-5G	267.24	0.0175	50	0.875	11.7
38	Cytidine	C9H13N3O5	nucleoside	Sigma	C4654-5G	243.22	0.0175	50	0.875	10.6
39	Cytosine	C4H5N3O	nucleobase	Sigma	C3506-1G	111.1	0.0175	50	0.875	4.9
40	Guanine	C5H5N5O	nucleobase	Sigma	G11950-100G	151.13	0.0175	50	0.875	6.6
41	Guanosine	C10H13N5O5	nucleoside	Sigma	G6264-5G	283.24	0.0175	50	0.875	12.4
42	Hypoxanthine	C5H4N4O	nucleobase	Sigma	H9636-5G	136.11	0.0175	50	0.875	6.0
43	Inosine	C10H12N4O5	nucleoside	Sigma	I4125-100G	268.23	0.0175	50	0.875	11.7
44	Thymine	C5H6N2O2	nucleobase	Sigma	T0895-5G	126.11	0.0175	50	0.875	5.5
45	Uracil	C4H4N2O2	nucleobase	Sigma	U1128-25G	112.09	0.0175	50	0.875	4.9
46	Uridine	C9H12N2O6	nucleoside	Sigma	U3003-5G	244.2	0.0175	50	0.875	10.7
47	Xanthine	C5H4N4O2	nucleobase	Sigma	X0626-5G	152.11	0.0175	50	0.875	6.7
48	Xanthosine dihydrate	C10H12N4O6	nucleobase	Sigma	X0750-5G	320.26	0.0175	50	0.875	14.0
NOTE: Dissolve all nucleobases and nucelosides in 0.08M NaOH

NLDM Solution 5 - 'Other_Cpds' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg for 50 mL stock solution
49	gamma-Aminobutyric acid	C4H9NO2	other	Sigma	A2129-25G	103.12	0.0175	50	0.875	4.5
50	Ectoine	C6H10N2O2	other	Sigma	81619-1G-F	142.16	0.0175	50	0.875	6.2
51	Betaine	C5H11NO2	amino acid deriv	Sigma	61962-50G	117.15	0.0175	50	0.875	5.1
52	N-acetyl-L-Glutamic acid	C7H11NO5	amino acid deriv	Sigma	855642-25G	189.17	0.0175	50	0.875	8.3
53	Nicotinamide	C6H6N2O	vitamin	Sigma	72340-100G	122.12	0.0175	50	0.875	5.3
54	Ornithine HCl	C5H12N2O2	amino acid deriv	Sigma	O2375-5G	168.62	0.0175	50	0.875	7.4
55	Shikimic acid	C7H10O5	other	Sigma	S5375-1G	174.15	0.0175	50	0.875	7.6
56	spermidine	C7H19N3	other	Sigma	S0266-1G	145.25	0.0175	50	0.875	6.4
57	taurine	C2H7NO3S	other	Sigma	T0625-10G	125.15	0.0175	50	0.875	5.5
58	Trigonelline HCl	C7H7NO2 x HCl	other	Sigma	T5509-5G	173.6	0.0175	50	0.875	7.6
59	L-Carnitine HCl	C7H16NO3 x HCl	other	Sigma	C0283-1G	197.66	0.0175	50	0.875	8.6
60	Choline Cl	C5H14NO	other	Sigma	C7017-5G	139.62	0.0175	50	0.875	6.1
61	N-acetyl-L-Glutamine	C7H12N2O4	amino acid deriv	Sigma	A9125-25G	188.18	0.0175	50	0.875	8.2
62	n-acetyl-L-lysine	C8H16N2O3	amino acid deriv	Sigma	A4021-1G	188.22	0.0175	50	0.875	8.2
63	n-acetylmuramic acid	C11H19NO8	other	Sigma	A3007-500MG	293.27	0.0175	50	0.875	12.8
64	sn-glycero-3-phosphocholine	C8H20NO6P	other	Cayman	20736	257.22	0.0175	50	0.875	11.3

A	B	C	D	E	F	G	H	I	J	K
NLDM Salt solution 1 - 'MgSO4' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg/50ml
S1	Magnesium sulfate heptahydrate	MgSO4 * 7H2O	Salt	Sigma	M2773-500G	246.47	0.812	50	40.6	500.3

NLDM Salt solution 2 - 'NH4Cl' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg/50ml
S2	Ammonium chloride	NH4Cl	Salt	Sigma	A9434-500G	53.49	1	50	50	133.7

NLDM Salt solution 3 - 'CaCl2' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg/50ml
S3	Calcium chloride dihydrate	CaCl2 * 2 H2O	Salt	Sigma	C7902-500G	147.01	0.68	50	34	249.9

NLDM Salt solution 4 - 'PO4' - 50X
#	Compound	formula	class	Supplier	Supplier #	MW (avg)	Final_Conc_mmol/L	Conc_Factor	Conc mmol/L	mg/50ml
S4	Sodium phosphate monobasic	NaH2PO4	Salt	Sigma	S0751-500G	119.98	1.665	50	83.25	499.4
S5	Potassium phosphate dibasic	K2HPO4	Salt	Sigma	P3786-500G	174.18	3.33	50	166.5	1450.0

	A
	Medium components
	NLDM Solution 1 - 'Sugars' - 10X
	NLDM Solution 2 - 'Organic_Acids' - 10X
	NLDM Solution 3 - 'Amino_Acids' - 10X
	NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X
	NLDM Solution 5 - 'Other_Cpds' - 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

	A	B
		Volume (mL)
	NLDM Solution 1 - 'Sugars' - 10X	50
	NLDM Solution 2 - 'Organic_Acids' - 10X	50
	NLDM Solution 3 - 'Amino_Acids' - 10X	50
	NLDM Solution 4 - 'Nucleobases/Nucleosides' - 50X	10
	NLDM Solution 5 - 'Other_Cpds' - 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
	Milli-Q Water	280
	TOTAL	500

Public workspaceNLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria

NLDM: A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria