#METABOLOMICS WORKBENCH fran_puig_20211126_083023 DATATRACK_ID:2947 STUDY_ID:ST002004 ANALYSIS_ID:AN003267 PROJECT_ID:PR001270 VERSION 1 CREATED_ON November 27, 2021, 8:57 am #PROJECT PR:PROJECT_TITLE A longitudinal study of the effect of temperature modification in full-scale PR:PROJECT_TITLE anaerobic digesters PR:PROJECT_SUMMARY In order to identify metabolites descriptive of alterations of the working PR:PROJECT_SUMMARY themperature during the process of anaerobic digestion, we performed untargeted PR:PROJECT_SUMMARY metabolomics on samples of sewage sludge collected from two reactors working in PR:PROJECT_SUMMARY parallel but with different temperature settings. PR:INSTITUTE INRAE PR:DEPARTMENT Microbiology and the food chain PR:LABORATORY PRocédés biOtechnologiques au Service de l'Environnement PR:LAST_NAME Chapleur PR:FIRST_NAME Olivier PR:ADDRESS 1 rue Pierre-Gilles de Gennes, 92761 Antony Cedex, FRANCE PR:EMAIL olivier.chapleur@inrae.fr PR:PHONE +33 0140966506 PR:FUNDING_SOURCE ANR #STUDY ST:STUDY_TITLE Metabolomics analysis of anaerobic digesters ST:STUDY_SUMMARY In order to identify metabolites descriptive of alterations of the working ST:STUDY_SUMMARY themperature during the process of anaerobic digestion, we performed untargeted ST:STUDY_SUMMARY metabolomics on samples of sewage sludge collected from two reactors working in ST:STUDY_SUMMARY parallel but with different temperature settings. ST:INSTITUTE INRAE ST:LAST_NAME Chapleur ST:FIRST_NAME Olivier ST:ADDRESS 1 rue Pierre-Gilles de Gennes, 92761 Antony Cedex, FRANCE ST:EMAIL olivier.chapleur@inrae.fr ST:PHONE +33 0140966506 ST:NUM_GROUPS 14 #SUBJECT SU:SUBJECT_TYPE Other abiotic sample SU:SUBJECT_SPECIES None #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 - a01 Reactor:A | Day:- RAW_FILE_NAME=a01; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a02 Reactor:B | Day:- RAW_FILE_NAME=a02; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a03 Reactor:A | Day:218 RAW_FILE_NAME=a03; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a04 Reactor:B | Day:218 RAW_FILE_NAME=a04; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - a05 Reactor:A | Day:353 RAW_FILE_NAME=a05; ExperimentDescription=full-digester; Temperature=32 SUBJECT_SAMPLE_FACTORS - a06 Reactor:B | Day:353 RAW_FILE_NAME=a06; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - a07 Reactor:A | Day:80 RAW_FILE_NAME=a07; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a08 Reactor:B | Day:80 RAW_FILE_NAME=a08; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a09 Reactor:A | Day:177 RAW_FILE_NAME=a09; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a10 Reactor:B | Day:177 RAW_FILE_NAME=a10; ExperimentDescription=full-digester; Temperature=30 SUBJECT_SAMPLE_FACTORS - a11 Reactor:A | Day:281 RAW_FILE_NAME=a11; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a12 Reactor:B | Day:281 RAW_FILE_NAME=a12; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - a13 Reactor:A | Day:462 RAW_FILE_NAME=a13; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - a14 Reactor:B | Day:462 RAW_FILE_NAME=a14; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b01 Reactor:A | Day:- RAW_FILE_NAME=b01; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b02 Reactor:B | Day:- RAW_FILE_NAME=b02; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b03 Reactor:A | Day:218 RAW_FILE_NAME=b03; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b04 Reactor:B | Day:218 RAW_FILE_NAME=b04; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - b05 Reactor:A | Day:353 RAW_FILE_NAME=b05; ExperimentDescription=full-digester; Temperature=32 SUBJECT_SAMPLE_FACTORS - b06 Reactor:B | Day:353 RAW_FILE_NAME=b06; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - b07 Reactor:A | Day:80 RAW_FILE_NAME=b07; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b08 Reactor:B | Day:80 RAW_FILE_NAME=b08; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b09 Reactor:A | Day:177 RAW_FILE_NAME=b09; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b10 Reactor:B | Day:177 RAW_FILE_NAME=b10; ExperimentDescription=full-digester; Temperature=30 SUBJECT_SAMPLE_FACTORS - b11 Reactor:A | Day:281 RAW_FILE_NAME=b11; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b12 Reactor:B | Day:281 RAW_FILE_NAME=b12; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - b13 Reactor:A | Day:462 RAW_FILE_NAME=b13; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - b14 Reactor:B | Day:462 RAW_FILE_NAME=b14; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200603195159 Reactor:- | Day:- RAW_FILE_NAME=blank_200603195159; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200603235248 Reactor:- | Day:- RAW_FILE_NAME=blank_200603235248; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200604045400 Reactor:- | Day:- RAW_FILE_NAME=blank_200604045400; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200604095509 Reactor:- | Day:- RAW_FILE_NAME=blank_200604095509; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200604145613 Reactor:- | Day:- RAW_FILE_NAME=blank_200604145613; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200604195714 Reactor:- | Day:- RAW_FILE_NAME=blank_200604195714; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200605005825 Reactor:- | Day:- RAW_FILE_NAME=blank_200605005825; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200605055939 Reactor:- | Day:- RAW_FILE_NAME=blank_200605055939; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200605090020 Reactor:- | Day:- RAW_FILE_NAME=blank_200605090020; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank_200605100030 Reactor:- | Day:- RAW_FILE_NAME=blank_200605100030; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - blank Reactor:- | Day:- RAW_FILE_NAME=blank; ExperimentDescription=blank; Temperature=35 SUBJECT_SAMPLE_FACTORS - c01 Reactor:A | Day:- RAW_FILE_NAME=c01; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c02 Reactor:B | Day:- RAW_FILE_NAME=c02; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c03 Reactor:A | Day:218 RAW_FILE_NAME=c03; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c04 Reactor:B | Day:218 RAW_FILE_NAME=c04; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - c05 Reactor:A | Day:353 RAW_FILE_NAME=c05; ExperimentDescription=full-digester; Temperature=32 SUBJECT_SAMPLE_FACTORS - c06 Reactor:B | Day:353 RAW_FILE_NAME=c06; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - c07 Reactor:A | Day:80 RAW_FILE_NAME=c07; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c08 Reactor:B | Day:80 RAW_FILE_NAME=c08; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c09 Reactor:A | Day:177 RAW_FILE_NAME=c09; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c10 Reactor:B | Day:177 RAW_FILE_NAME=c10; ExperimentDescription=full-digester; Temperature=30 SUBJECT_SAMPLE_FACTORS - c11 Reactor:A | Day:281 RAW_FILE_NAME=c11; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c12 Reactor:B | Day:281 RAW_FILE_NAME=c12; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - c13 Reactor:A | Day:462 RAW_FILE_NAME=c13; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - c14 Reactor:B | Day:462 RAW_FILE_NAME=c14; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d01 Reactor:A | Day:- RAW_FILE_NAME=d01; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d02 Reactor:B | Day:- RAW_FILE_NAME=d02; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d03 Reactor:A | Day:218 RAW_FILE_NAME=d03; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d04 Reactor:B | Day:218 RAW_FILE_NAME=d04; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - d05 Reactor:A | Day:353 RAW_FILE_NAME=d05; ExperimentDescription=full-digester; Temperature=32 SUBJECT_SAMPLE_FACTORS - d06 Reactor:B | Day:353 RAW_FILE_NAME=d06; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - d07 Reactor:A | Day:80 RAW_FILE_NAME=d07; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d08 Reactor:B | Day:80 RAW_FILE_NAME=d08; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d09 Reactor:A | Day:177 RAW_FILE_NAME=d09; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d10 Reactor:B | Day:177 RAW_FILE_NAME=d10; ExperimentDescription=full-digester; Temperature=30 SUBJECT_SAMPLE_FACTORS - d11 Reactor:A | Day:281 RAW_FILE_NAME=d11; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d12 Reactor:B | Day:281 RAW_FILE_NAME=d12; ExperimentDescription=full-digester; Temperature=25 SUBJECT_SAMPLE_FACTORS - d13 Reactor:A | Day:462 RAW_FILE_NAME=d13; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - d14 Reactor:B | Day:462 RAW_FILE_NAME=d14; ExperimentDescription=full-digester; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool08 Reactor:- | Day:- RAW_FILE_NAME=pool08; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool09 Reactor:- | Day:- RAW_FILE_NAME=pool09; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool1 Reactor:- | Day:- RAW_FILE_NAME=pool1; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool2 Reactor:- | Day:- RAW_FILE_NAME=pool2; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool3 Reactor:- | Day:- RAW_FILE_NAME=pool3; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool4 Reactor:- | Day:- RAW_FILE_NAME=pool4; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool5 Reactor:- | Day:- RAW_FILE_NAME=pool5; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool6 Reactor:- | Day:- RAW_FILE_NAME=pool6; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool7 Reactor:- | Day:- RAW_FILE_NAME=pool7; ExperimentDescription=pool; Temperature=35 SUBJECT_SAMPLE_FACTORS - pool Reactor:- | Day:- RAW_FILE_NAME=pool; ExperimentDescription=pool; Temperature=35 #COLLECTION CO:COLLECTION_SUMMARY Sludge samples from two reactors were collected at days 0, 80, 177, 218, 281, CO:COLLECTION_SUMMARY 353, and 462. Sludge was collected in 50-mL sterile Falcon tubes and immediately CO:COLLECTION_SUMMARY transported on dry ice to INRAE, where they were frozen at -80 °C. CO:SAMPLE_TYPE Sewage sludge #TREATMENT TR:TREATMENT_SUMMARY During the time of the experiment, both reactors (3,000 m3 each) worked at half TR:TREATMENT_SUMMARY capacity due to a constraint in the availability of substrate to treat. On TR:TREATMENT_SUMMARY average, the full-scale digesters were operated at a mean hydraulic retention TR:TREATMENT_SUMMARY time (HRT) of 34 days and a mean organic loading rate of 0.82 kg of volatile TR:TREATMENT_SUMMARY solids m-3 day-1. For the first four months of study, the two reactors operated TR:TREATMENT_SUMMARY at 35 °C. Reactor A was kept at this temperature during all the experiment. For TR:TREATMENT_SUMMARY digester B, the temperature was decreased abruptly to 30 °C on the 123th day TR:TREATMENT_SUMMARY and maintained at this level for 2 HRTs until the 190th day. Then, the TR:TREATMENT_SUMMARY temperature was modified to 25 °C and maintained for 3 HRTs (between the 191st TR:TREATMENT_SUMMARY and the 373th days). Finally, the heating of reactor B was increased from day TR:TREATMENT_SUMMARY 374 onwards to reach the initial temperature of 35 °C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For every sample, four aliquots with 0.75 mL of sludge were prepared in 2-mL SP:SAMPLEPREP_SUMMARY Eppendorf tubes. Then, 1 mL of phosphate buffer saline was added into each tube SP:SAMPLEPREP_SUMMARY and the sludge volumes were washed by centrifuging them at 15500 rcf for 3 min SP:SAMPLEPREP_SUMMARY at 4 °C. This process was repeated once. The samples were freeze-dried SP:SAMPLEPREP_SUMMARY overnight and, for every sample, 20 mg of dried sludge were transferred in new SP:SAMPLEPREP_SUMMARY Eppendorf tubes. Then, the dried sludge powders were resuspended in 1 mL of a SP:SAMPLEPREP_SUMMARY cold ddH2O: MeOH mixture (1:1) containing 2.5 mM PIPES. The samples were SP:SAMPLEPREP_SUMMARY sonicated for 6 min, and submerged in liquid nitrogen. After one minute, the SP:SAMPLEPREP_SUMMARY samples were thawed in ice. The cold-shock step was repeated twice. Afterwards, SP:SAMPLEPREP_SUMMARY the samples were agitated for 15 min at 420 rpm in an orbital shaker. Following SP:SAMPLEPREP_SUMMARY this, the samples were centrifuged at 15500 rcf for 3 min at 4 °C and the SP:SAMPLEPREP_SUMMARY supernatants were collected. The supernatants were immediately filtered with SP:SAMPLEPREP_SUMMARY 0.45 μM Nylon filters and freeze-dried overnight. The dried extracts were left SP:SAMPLEPREP_SUMMARY at -80°C until the HPLC-MS analyses. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Accela 1250 CH:COLUMN_NAME Macherey Nagel Nucleodur HILIC (100 x 2 mm, 1.8 µm) CH:FLOW_GRADIENT 95% B: 3 min, 95% - 60% B: 3 - 14 min, 60% - 95 % B: 14 -24 min, 95% B: 24 - 29 CH:FLOW_GRADIENT min CH:FLOW_RATE 0.4 mL/min CH:SOLVENT_A Ammonium acetate 50 Mm at pH 5 CH:SOLVENT_B Acetonitrile CH:SAMPLE_INJECTION 10 µL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo LTQ XL MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS XCMS online for assignment METLIN, HMDB MS:CAPILLARY_TEMPERATURE 320 °C MS:MS_RESULTS_FILE ST002004_AN003267_Results.txt UNITS:Peak areas Has m/z:Yes Has RT:Yes RT units:Minutes #END