#METABOLOMICS WORKBENCH ghiwa_makke_20250331_112044 DATATRACK_ID:5809 STUDY_ID:ST003907 ANALYSIS_ID:AN006412 PROJECT_ID:PR002445 VERSION 1 CREATED_ON May 8, 2025, 12:41 pm #PROJECT PR:PROJECT_TITLE Northern peatland microbial communities exhibit resistance to warming and PR:PROJECT_TITLE acquire electron acceptors from soil organic matter PR:PROJECT_SUMMARY The microbial networks that regulate belowground carbon turnover and respond to PR:PROJECT_SUMMARY climate change drivers in peatlands are poorly understood. Here, we leverage a PR:PROJECT_SUMMARY whole ecosystem warming experiment to elucidate the key processes of terminal PR:PROJECT_SUMMARY carbon decomposition and community responses to temperature rise. Our dataset of PR:PROJECT_SUMMARY 697 metagenome-assembled genomes (MAGs) extends from surface (10 cm) to 2 m deep PR:PROJECT_SUMMARY into the peat column, with only 3.7% of genomes overlapping with other PR:PROJECT_SUMMARY well-studied peatlands. Unexpectedly, community composition has yet to show a PR:PROJECT_SUMMARY significant response to warming after 3 years, suggesting that metabolically PR:PROJECT_SUMMARY diverse soil microbial networks are resilient to climate change. Surprisingly, PR:PROJECT_SUMMARY the dominant methanogens showed the potential for both acetoclastic and PR:PROJECT_SUMMARY hydrogenotrophic methanogenesis. Nonetheless, the predominant pathways for PR:PROJECT_SUMMARY anaerobic carbon decomposition include sulfate/sulfite reduction, PR:PROJECT_SUMMARY denitrification, and acetogenesis, rather than methanogenesis based on gene PR:PROJECT_SUMMARY abundances. Multi-omics data suggest that organic matter cleavage provides PR:PROJECT_SUMMARY terminal electron acceptors, which together with methanogen metabolic PR:PROJECT_SUMMARY flexibility, may explain peat microbiome resilience to warming. PR:INSTITUTE University of Arizona PR:DEPARTMENT Environmental Science PR:LABORATORY Tfaily Lab PR:LAST_NAME Makke PR:FIRST_NAME Ghiwa PR:ADDRESS 1230 North Cherry Avenue, Tucson, AZ, 85721, USA PR:EMAIL ghiwamakke@arizona.edu PR:PHONE 520-626-3650 #STUDY ST:STUDY_TITLE Northern peatland microbial networks exhibit resilience to warming and acquire ST:STUDY_TITLE electron acceptor from soil organic matter ST:STUDY_SUMMARY Peatlands store vast amounts of carbon, but how their microbial and chemical ST:STUDY_SUMMARY processes respond to climate change remains unclear. In this study, we ST:STUDY_SUMMARY investigated how soil microbial communities and organic matter chemistry in a ST:STUDY_SUMMARY northern Minnesota peatland respond to long-term experimental warming. Peat ST:STUDY_SUMMARY samples were collected from four depths (10–175 cm) across 10 whole-ecosystem ST:STUDY_SUMMARY warming enclosures at the SPRUCE (Spruce and Peatland Responses Under Changing ST:STUDY_SUMMARY Environments) site in the Marcell Experimental Forest. We used liquid ST:STUDY_SUMMARY chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the chemical ST:STUDY_SUMMARY composition of peat at different depths, alongside metagenomic sequencing to ST:STUDY_SUMMARY reconstruct nearly 700 microbial genomes. Despite sustained warming for three ST:STUDY_SUMMARY years, microbial community structure remained stable, indicating resilience to ST:STUDY_SUMMARY temperature stress. However, functional gene analysis and metabolomics revealed ST:STUDY_SUMMARY shifts in carbon processing pathways, with sulfate reduction, denitrification, ST:STUDY_SUMMARY and acetogenesis emerging as key decomposition processes. Genome-resolved ST:STUDY_SUMMARY metagenomics further revealed methanogen metabolic flexibility, including ST:STUDY_SUMMARY potential for hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis ST:STUDY_SUMMARY pathways. Supporting this, integration of metabolomic and genomic data ST:STUDY_SUMMARY highlighted a key mechanism by which microbial metabolism may access alternative ST:STUDY_SUMMARY electron acceptors: the ratio of choline-O-sulfate to choline was strongly ST:STUDY_SUMMARY correlated with the abundance of the betC gene, which encodes choline sulfatase. ST:STUDY_SUMMARY This enzyme cleaves choline-O-sulfate to release sulfate, potentially fueling ST:STUDY_SUMMARY anaerobic respiration via sulfate-reducing microbes. These findings suggest that ST:STUDY_SUMMARY microbial metabolic flexibility, coupled with changes in peat chemistry, plays a ST:STUDY_SUMMARY critical role in maintaining peatland carbon cycling under warming conditions. ST:INSTITUTE University of Arizona ST:DEPARTMENT Environmental Science ST:LABORATORY Tfaily Lab ST:LAST_NAME Makke ST:FIRST_NAME Ghiwa ST:ADDRESS 1230 North Cherry Avenue ST:EMAIL ghiwamakke@arizona.edu ST:PHONE 5209106052 #SUBJECT SU:SUBJECT_TYPE Soil sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - P4-20-2018 Sample source:Soil | Depth:D20 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P4-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P4-20-2018.raw SUBJECT_SAMPLE_FACTORS - P4-40-2018 Sample source:Soil | Depth:D40 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P4-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P4-40-2018.raw SUBJECT_SAMPLE_FACTORS - P4-100-2018 Sample source:Soil | Depth:D100 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P4-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P4-100-2018.raw SUBJECT_SAMPLE_FACTORS - P4-150-2018 Sample source:Soil | Depth:D150 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P4-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P4-150-2018.raw SUBJECT_SAMPLE_FACTORS - P6-20-2018 Sample source:Soil | Depth:D20 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P6-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P6-20-2018.raw SUBJECT_SAMPLE_FACTORS - P6-40-2018 Sample source:Soil | Depth:D40 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P6-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P6-40-2018.raw SUBJECT_SAMPLE_FACTORS - P6-100-2018 Sample source:Soil | Depth:D100 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P6-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P6-100-2018.raw SUBJECT_SAMPLE_FACTORS - P6-150-2018 Sample source:Soil | Depth:D150 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P6-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P6-150-2018.raw SUBJECT_SAMPLE_FACTORS - P8-20-2018 Sample source:Soil | Depth:D20 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P8-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P8-20-2018.raw SUBJECT_SAMPLE_FACTORS - P8-40-2018 Sample source:Soil | Depth:D40 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P8-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P8-40-2018.raw SUBJECT_SAMPLE_FACTORS - P8-100-2018 Sample source:Soil | Depth:D100 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P8-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P8-100-2018.raw SUBJECT_SAMPLE_FACTORS - P8-150-2018 Sample source:Soil | Depth:D150 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P8-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P8-150-2018.raw SUBJECT_SAMPLE_FACTORS - P10-20-2018 Sample source:Soil | Depth:D20 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P10-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P10-20-2018.raw SUBJECT_SAMPLE_FACTORS - P10-40-2018 Sample source:Soil | Depth:D40 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P10-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P10-40-2018.raw SUBJECT_SAMPLE_FACTORS - P10-100-2018 Sample source:Soil | Depth:D100 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P10-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P10-100-2018.raw SUBJECT_SAMPLE_FACTORS - P10-150-2018 Sample source:Soil | Depth:D150 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P10-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P10-150-2018.raw SUBJECT_SAMPLE_FACTORS - P11-20-2018 Sample source:Soil | Depth:D20 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P11-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P11-20-2018.raw SUBJECT_SAMPLE_FACTORS - P11-40-2018 Sample source:Soil | Depth:D40 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P11-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P11-40-2018.raw SUBJECT_SAMPLE_FACTORS - P11-100-2018 Sample source:Soil | Depth:D100 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P11-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P11-100-2018.raw SUBJECT_SAMPLE_FACTORS - P11-150-2018 Sample source:Soil | Depth:D150 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P11-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P11-150-2018.raw SUBJECT_SAMPLE_FACTORS - P13-20-2018 Sample source:Soil | Depth:D20 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P13-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P13-20-2018.raw SUBJECT_SAMPLE_FACTORS - P13-40-2018 Sample source:Soil | Depth:D40 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P13-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P13-40-2018.raw SUBJECT_SAMPLE_FACTORS - P13-100-2018 Sample source:Soil | Depth:D100 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P13-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P13-100-2018.raw SUBJECT_SAMPLE_FACTORS - P13-150-2018 Sample source:Soil | Depth:D150 | Temperature:4.5 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P13-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P13-150-2018.raw SUBJECT_SAMPLE_FACTORS - P16-20-2018 Sample source:Soil | Depth:D20 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P16-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P16-20-2018.raw SUBJECT_SAMPLE_FACTORS - P16-40-2018 Sample source:Soil | Depth:D40 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P16-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P16-40-2018.raw SUBJECT_SAMPLE_FACTORS - P16-100-2018 Sample source:Soil | Depth:D100 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P16-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P16-100-2018.raw SUBJECT_SAMPLE_FACTORS - P16-150-2018 Sample source:Soil | Depth:D150 | Temperature:6.75 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P16-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P16-150-2018.raw SUBJECT_SAMPLE_FACTORS - P17-20-2018 Sample source:Soil | Depth:D20 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P17-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P17-20-2018.raw SUBJECT_SAMPLE_FACTORS - P17-40-2018 Sample source:Soil | Depth:D40 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P17-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P17-40-2018.raw SUBJECT_SAMPLE_FACTORS - P17-100-2018 Sample source:Soil | Depth:D100 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P17-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P17-100-2018.raw SUBJECT_SAMPLE_FACTORS - P17-150-2018 Sample source:Soil | Depth:D150 | Temperature:9 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P17-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P17-150-2018.raw SUBJECT_SAMPLE_FACTORS - P19-20-2018 Sample source:Soil | Depth:D20 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P19-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P19-20-2018.raw SUBJECT_SAMPLE_FACTORS - P19-40-2018 Sample source:Soil | Depth:D40 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P19-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P19-40-2018.raw SUBJECT_SAMPLE_FACTORS - P19-100-2018 Sample source:Soil | Depth:D100 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P19-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P19-100-2018.raw SUBJECT_SAMPLE_FACTORS - P19-150-2018 Sample source:Soil | Depth:D150 | Temperature:0 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P19-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P19-150-2018.raw SUBJECT_SAMPLE_FACTORS - P20-20-2018 Sample source:Soil | Depth:D20 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P20-20-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P20-20-2018.raw SUBJECT_SAMPLE_FACTORS - P20-40-2018 Sample source:Soil | Depth:D40 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P20-40-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P20-40-2018.raw SUBJECT_SAMPLE_FACTORS - P20-100-2018 Sample source:Soil | Depth:D100 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P20-100-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P20-100-2018.raw SUBJECT_SAMPLE_FACTORS - P20-150-2018 Sample source:Soil | Depth:D150 | Temperature:2.25 RAW_FILE_NAME(Raw file name _RP)=RP_Pos_P20-150-2018.raw; RAW_FILE_NAME(Raw file name_HILIC)=HILIC_Neg_P20-150-2018.raw #COLLECTION CO:COLLECTION_SUMMARY Peat samples were collected in August of 2018 from the hollows of each of the 10 CO:COLLECTION_SUMMARY whole-ecosystem warming chambers of the Spruce and Peatland Responses Under CO:COLLECTION_SUMMARY Changing Environments (SPRUCE) warming experiment located in the ombrotrophic, CO:COLLECTION_SUMMARY acidic S1 Bog of the Marcell Experimental Forest, north of Grand Rapids, MN with CO:COLLECTION_SUMMARY a serrated knife at the surface and a Russian corer at depth. The samples were CO:COLLECTION_SUMMARY separated into depth increments and six 0.35-g subsamples of homogenized peat CO:COLLECTION_SUMMARY from the 10-20 cm, 40-50 cm, 100-125 cm, and 150-175 cm depth increments. CO:SAMPLE_TYPE soil #TREATMENT TR:TREATMENT_SUMMARY The SPRUCE experiment where the samples were collected consists of 17 open-top TR:TREATMENT_SUMMARY chambers that control the peat and air temperature (ambient, +0, +2.25, +4.5, TR:TREATMENT_SUMMARY +6.75 and +9°C) as well as atmospheric CO2 concentration (ambient and 900 ppm). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To dry samples and ensure uniform starting weight for extraction, peat samples SP:SAMPLEPREP_SUMMARY were first lyophilized using a Labconco FreeZone, Benchtop freeze dryer for 48 SP:SAMPLEPREP_SUMMARY hr. The freeze-dried peat samples (0.2 g) were extracted by adding 20 mL of an SP:SAMPLEPREP_SUMMARY 80:20 solution of MeOH: sterile MilliQ water. Samples were briefly vortexed and SP:SAMPLEPREP_SUMMARY sonicated in a water bath for 2 hr at 20 °C (FisherBrand CPX3800). The SP:SAMPLEPREP_SUMMARY supernatant was filtered through a 0.45 um filter to remove cellular debris and SP:SAMPLEPREP_SUMMARY plant material. Of this extract, 7 mL was transferred to two glass autosampler SP:SAMPLEPREP_SUMMARY vials (3.5 mL each), dried in a vacuum centrifuge (Eppendorf Vacufuge plus), and SP:SAMPLEPREP_SUMMARY stored at −80 °C. Prior to CL-MS/MS analysis, samples were reconstituted in SP:SAMPLEPREP_SUMMARY 80:20 water: methanol for reverse phase (RP), and 50:50 water: acetonitrile for SP:SAMPLEPREP_SUMMARY hydrophilic interaction liquid chromatography (HILIC). SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish UHPLC CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% Methanol; 0.1% formic acid CH:FLOW_GRADIENT 0–3 min held at 1% B; 3–19 min 1% B – 95% B; 19–20 min 95% B CH:FLOW_RATE 300 uL/minute CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Analytical & Biological Mass Spectrometry Core Facility (University of Arizona) #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data analysis was conducted using the Compound Discoverer 3.3 software by Thermo MS:MS_COMMENTS Fisher Scientific, employing an untargeted metabolomics workflow MS:MS_RESULTS_FILE ST003907_AN006412_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END