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Acetonitrile extracts from soil samples with added nitrification inhibitors MHPA, MBOA and Limonene

ABSTRACT: Measurement of DMPP, MBOA, Methyl p-coumerate (MHPA) and Limonene in soil extracts.

Nitrification inhibitors extraction protocols
BNIs extraction with acetonitrile:

Solutions:
Acetonitrile HPLC grade

Protocol:
1. Weight 2g of soil and add NH4+ solution and the BNIs as powder (MHPA, MBOA) or liquid (limonene)
2. Add 4mL acetonitrile
3. Shake the mixture vigorously by hand before being putting the centrifuge tubes horizontally on an orbital shaker for 30 min (200 rpm)
4. Centrifuge at 10000 rpm for 10mins
5. Filter supernatant through a 0.45um
6. Store -20C

DMPP extraction:
Solutions:
HPLC-eluent (212 ml methanol + 788 ml of a 1 mM H3PO4 and 5 mM KH2PO4 buffer
2 M KCl
1 M NaOH
di-ethyl-ether
Protocol:
1. Weigh 2 g of field moist soil into 50 mL tapered polypropylene centrifuge tube and add NH4 with DMPP solutions (100uL for Marchfeld, 30uL for Wieselburg)
2. Add 2 mL HPLC-eluent
3. Add 8 mL 2 M KCl
4. Shake the mixture vigorously by hand before being putting the centrifuge tubes horizontally on an orbital shaker for 60 min (170 rpm)
5. Centrifuge the extracts at 10000 rpm for 5 min. Decant and transfer to fresh 50mL centrifuge tube
6. Add 400uL NaOH + 8mL di-ethylether
7. Shake 10mins and then centrifuge at 10000rpm for 10mins
8. Transfer 8mL of upper di-ethylether in a 50mL falcon tube containing 2mL HPLC eluent
9. Evaporate di-ehtylether overnight
10. Transfer about 2mL into an eppendrof
11. Store -20C

Measurements according to 10.1021/ac503290j, but with an Q Exactive HF instrument

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Unidentified Soil Bacteria (ncbitaxon:34109)

SUBMITTER: christoph bueschl

PROVIDER: MSV000100563 | MassIVE | Fri Jan 23 01:51:00 GMT 2026

REPOSITORIES: MassIVE

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Project description:Nitrosomonas europaea Nm50 BNI degradation experiment - MBOA and D-DMPP - concentrate ~ 600 ml culture - resuspend to make mother-culture - 50 ml in 100 ml shot bottle. Feed 1 mM NH4Cl (final concentration) to mother culture bottle to keep active till 25th and setup - Keep at 28C overnight After two days - Wash remaining 25 ml of mother culture (2x 12.5 in one spin column 50 ml tube: use "spent/already used 2nd tube as Tara with 12.5 ml water, during centrifugation); wash 3x with medium (check if nitrite is gone, resuspend in 170 ml medium - need 160 for setup + mother culture samples) - let acclimate for ~20min - 40 min without substrate (lunch) - prepare 84 ml medium with 0.5 mM ammonium for abiotic controls - add 1 mM ammonium to mother culture, sample M0, M1 (20 min), M2 (40 min) D-DMPP mix (790% D, 30% notz labeled) dissolved in MQ to 0.05 M (50 mM). [17.95 mg D-DMPP + 5.3 mg DMPP in 2.4 mL MQ] Split mother culture into the following incubations, in small plastic vials I will give you: A: 5 x 8 ml culture; mother stock 42; no DMPP (just 40 ul medium) B: 5 X 8 ml culture mother stock 42 + 1 uM DMPP final concentration (2.1 ul emulsion) C: 5 X 8 ml culture mother stock 42 + 2.5 uM DMPP final concentration (21 ul emulsion) D: 5 x 8 ml culture mother stock 42 + 10 uM DMPP final concentration (210 ul emulsion) ABIOTICS: E: 5x 8 ml medium (with 0.5 ammonium); mother stock 42 + 1 uM DMPP final concentration (2.1 ul emulsion) F: 5x 8 ml medium (with 0.5 ammonium) mother stock 42 + 10 uM DMPPfinal concentration (210 ul emulsion) Take T0 Sample every 20-30-60 min; 0.5 ml for N assay and 0.5 ml for analytics (for E and F only T0, T2, T4) Sample preparation d-DMPP samples: 1.Centrifuge samples in Eppendorf tubes at 30,000 g for 10 minutes at 4C 2.Transfer 300 uL supernatant into HPLC vials with 300/400 uL inserts Measurements according to 10.1021/ac503290j, but with an Q Exactive HF instrument

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Acetonitrile extracts from soil samples with added nitrification inhibitors MHPA, MBOA and Limonene

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