#METABOLOMICS WORKBENCH deshpa11_20240307_131037 DATATRACK_ID:4692 STUDY_ID:ST003119 ANALYSIS_ID:AN005113
VERSION                          	1
CREATED_ON                       	04-01-2024
#PROJECT
PR:PROJECT_TITLE                 	Bleach protects algal crops from novel pathogen but increases stress tolerance
PR:PROJECT_TITLE                 	traits and virulence factors within the associated bacterial microbiome
PR:PROJECT_SUMMARY               	Analysis of exometabolites in algal ponds cultivated with or without bleach
PR:PROJECT_SUMMARY               	treatment for pest management. The goal of this work is to uncover metabolic
PR:PROJECT_SUMMARY               	features that drive differences in bleach treated and unbleached algal
PR:PROJECT_SUMMARY               	cultivation ponds. Six ponds were used for the experiment, with three ponds
PR:PROJECT_SUMMARY               	managed for pests via sodium hypochlorite (ponds C2, C3, and C4), and three
PR:PROJECT_SUMMARY               	ponds unmanaged (A3, B2, and B4) and samples were taken at 7 different
PR:PROJECT_SUMMARY               	timepoints (1-7) from early growth phase at day 1 to day 16.
PR:INSTITUTE                     	National Renewable Energy Lab (NREL)
PR:DEPARTMENT                    	Bioscience
PR:LABORATORY                    	Algae Lab
PR:LAST_NAME                     	Deshpande
PR:FIRST_NAME                    	Arnav
PR:ADDRESS                       	15013 Denver West Parkway
PR:EMAIL                         	arnav.deshpande@nrel.gov
PR:PHONE                         	7657759029
PR:DOI                           	http://dx.doi.org/10.21228/M8BM7V
#STUDY
ST:STUDY_TITLE                   	Bleach protects algal crops from novel pathogen but increases stress tolerance
ST:STUDY_TITLE                   	traits and virulence factors within the associated bacterial microbiome
ST:STUDY_SUMMARY                 	Microalgae farming is a promising and growing industry. Most large-scale
ST:STUDY_SUMMARY                 	cultivation is done in open pond or raceway systems, as other approaches (e.g.,
ST:STUDY_SUMMARY                 	photobioreactors) have high upfront capital costs. Open pond cultivation systems
ST:STUDY_SUMMARY                 	leave cultures vulnerable to contamination by pests including rotifers,
ST:STUDY_SUMMARY                 	ciliates, and flagellates that feed on microalgae. Pest organisms can either
ST:STUDY_SUMMARY                 	decrease algal productivity or lead to wholesale crop loss (i.e., a pond crash)
ST:STUDY_SUMMARY                 	which can significantly lower annual production rates and increase annual costs.
ST:STUDY_SUMMARY                 	Sodium hypochlorite is the active ingredient in bleach, an effective and
ST:STUDY_SUMMARY                 	inexpensive chlorine-based disinfectant commonly used at scale to protect algal
ST:STUDY_SUMMARY                 	crops. In this study, we investigate 1) if and how bleach treatment changes
ST:STUDY_SUMMARY                 	bacterial community composition, 2) if changes to bacterial composition result
ST:STUDY_SUMMARY                 	in changes in the functional potential of the bacterial community, 3) if an
ST:STUDY_SUMMARY                 	algal pathogen can be identified as the cause of pond crashes in untreated
ST:STUDY_SUMMARY                 	ponds, and finally, 4) explore the effect of bleach treatment on metabolic
ST:STUDY_SUMMARY                 	features present in bleached and unbleached ponds, 5) explore the effect of
ST:STUDY_SUMMARY                 	treatment on virulence factors associated with known human and animal pathogens.
ST:INSTITUTE                     	National Renewable Energy Lab (NREL)
ST:LAST_NAME                     	Deshpande
ST:FIRST_NAME                    	Arnav
ST:ADDRESS                       	15013 Denver West Parkway
ST:EMAIL                         	arnav.deshpande@nrel.gov
ST:PHONE                         	7657759029
ST:SUBMIT_DATE                   	2024-03-07
#SUBJECT
SU:SUBJECT_TYPE                  	Water sample
SU:SUBJECT_SPECIES               	Algae
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	S_1	Treatment:Bleached | Timepoint:1 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_2	Treatment:Bleached | Timepoint:1 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_3	Treatment:Bleached | Timepoint:1 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_4	Treatment:Bleached | Timepoint:2 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_4.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_5	Treatment:Bleached | Timepoint:2 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_5.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_6	Treatment:Bleached | Timepoint:2 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_6.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_7	Treatment:Bleached | Timepoint:3 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_7.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_8	Treatment:Bleached | Timepoint:3 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_8.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_9	Treatment:Bleached | Timepoint:3 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_9.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_10	Treatment:Bleached | Timepoint:4 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_10.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_11	Treatment:Bleached | Timepoint:4 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_11.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_12	Treatment:Bleached | Timepoint:4 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_12.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_13	Treatment:Bleached | Timepoint:5 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_13.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_14	Treatment:Bleached | Timepoint:5 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_14.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_15	Treatment:Bleached | Timepoint:5 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_15.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_16	Treatment:Bleached | Timepoint:6 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_16.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_17	Treatment:Bleached | Timepoint:6 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_17.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_18	Treatment:Bleached | Timepoint:6 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_18.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_19	Treatment:Bleached | Timepoint:7 | Sample source:Pond C2	RAW_FILE_NAME(Raw file name)=S_19.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_20	Treatment:Bleached | Timepoint:7 | Sample source:Pond C3	RAW_FILE_NAME(Raw file name)=S_20.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_21	Treatment:Bleached | Timepoint:7 | Sample source:Pond C4	RAW_FILE_NAME(Raw file name)=S_21.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_22	Treatment:Unbleached | Timepoint:1 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_22.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_23	Treatment:Unbleached | Timepoint:1 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_23.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_24	Treatment:Unbleached | Timepoint:1 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_24.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_25	Treatment:Unbleached | Timepoint:2 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_25.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_26	Treatment:Unbleached | Timepoint:2 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_26.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_27	Treatment:Unbleached | Timepoint:2 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_27.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_28	Treatment:Unbleached | Timepoint:3 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_28.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_29	Treatment:Unbleached | Timepoint:3 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_29.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_30	Treatment:Unbleached | Timepoint:3 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_30.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_31	Treatment:Unbleached | Timepoint:4 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_31.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_32	Treatment:Unbleached | Timepoint:4 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_32.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_33	Treatment:Unbleached | Timepoint:4 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_33.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_34	Treatment:Unbleached | Timepoint:5 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_34.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_35	Treatment:Unbleached | Timepoint:5 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_35.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_36	Treatment:Unbleached | Timepoint:5 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_36.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_37	Treatment:Unbleached | Timepoint:6 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_37.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_38	Treatment:Unbleached | Timepoint:6 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_38.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_39	Treatment:Unbleached | Timepoint:6 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_39.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_40	Treatment:Unbleached | Timepoint:7 | Sample source:Pond A3	RAW_FILE_NAME(Raw file name)=S_40.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_41	Treatment:Unbleached | Timepoint:7 | Sample source:Pond B2	RAW_FILE_NAME(Raw file name)=S_41.mzML
SUBJECT_SAMPLE_FACTORS           	-	S_42	Treatment:Unbleached | Timepoint:7 | Sample source:Pond B4	RAW_FILE_NAME(Raw file name)=S_42.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Samples were collected from all 6 ponds (bleached and unbleached) at 7 different
CO:COLLECTION_SUMMARY            	times during algal growth. The algae was separated by centrifugation at 3700 rpm
CO:COLLECTION_SUMMARY            	for 10 minutes. The supernatant was filtered through a 0.22μm filter.
CO:SAMPLE_TYPE                   	Culture Media
CO:COLLECTION_LOCATION           	New Mexico State University, Las Cruces
CO:COLLECTION_FREQUENCY          	7 times over a 16 day culture period
CO:STORAGE_CONDITIONS            	-20℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Six ponds were used for the experiment, with three ponds managed for pests via
TR:TREATMENT_SUMMARY             	sodium hypochlorite (ponds C2, C3, and C4), and three ponds unmanaged (A3, B2,
TR:TREATMENT_SUMMARY             	and B4). The experiment was started on 9/27/2022 when the optical density at a
TR:TREATMENT_SUMMARY             	wavelength of 750 nanometers (OD750) was measured at 0.5 for each pond and
TR:TREATMENT_SUMMARY             	lasted for 16 days, concluding on 10/13/2022. Bleach was added to ponds C2, C3,
TR:TREATMENT_SUMMARY             	and C4 on 09/30/22, 10/04/22, 10/07/22, and 10/11/22 at 15 ppm.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Algae separated by centrifugation at 3700 rpm for 10 mins followed by filtration
SP:SAMPLEPREP_SUMMARY            	using a 0.22μm filter
#CHROMATOGRAPHY
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
CH:COLUMN_TEMPERATURE            	25°C
CH:FLOW_GRADIENT                 	90% solvent B for 1 min followed by a linear gradient down to 75% solvent B for
CH:FLOW_GRADIENT                 	23 min, a linear gradient down to 45% solvent B in 2 min, a linear gradient down
CH:FLOW_GRADIENT                 	to 25% B in 4 min, followed by a step to the starting composition of 90% B and
CH:FLOW_GRADIENT                 	hold for 6 min for column regeneration for a total run time of 36 min.
CH:FLOW_RATE                     	0.2 mL/min
CH:INTERNAL_STANDARD             	1-13C Leucine
CH:SAMPLE_INJECTION              	20 µL
CH:SOLVENT_A                     	97% 18.2 mΩ-cm water/3% acetonitrile; 20 mM ammonium acetate; 15 mM ammonium
CH:SOLVENT_A                     	hydroxide
CH:SOLVENT_B                     	95% acetonitrile/5% 18.2 mΩ-cm water; 20 mM ammonium acetate; 15 mM ammonium
CH:SOLVENT_B                     	hydroxide
CH:CHROMATOGRAPHY_TYPE           	HILIC
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Mass spectrometer data was collected using a Thermo Scientific Q-Exactive mass
MS:MS_COMMENTS                   	spectrometer in negative ion mode. The scan range was from 60 to 900 m/z with a
MS:MS_COMMENTS                   	resolution of 140,000. Data was collected only at the MS1 scan level. Untargeted
MS:MS_COMMENTS                   	metabolomics was performed using Compound Discoverer 3.2 (Thermo Fisher
MS:MS_COMMENTS                   	Scientific). Retention time tolerance was set to 0.15 min. The detect compounds
MS:MS_COMMENTS                   	node was used with 5 ppm mass tolerance, an S/N threshold of 6, 50000 minimum
MS:MS_COMMENTS                   	peak intensity. The maximum peak width was set to 0.5 min, with a minimum 5
MS:MS_COMMENTS                   	scans per peak and a minimum of 2 isotopes detected. Only the compounds with
MS:MS_COMMENTS                   	[M-H]-1 were selected for detection. Compounds were grouped with retention time
MS:MS_COMMENTS                   	(RT) tolerance of 0.2 min. The Fill Gaps node was used with 5 ppm mass
MS:MS_COMMENTS                   	tolerance, missing value imputation was implemented with random forest settings
MS:MS_COMMENTS                   	with number of trees set to 100 and max iterations set to 10. Background
MS:MS_COMMENTS                   	features were removed with max sample/blank set to 5. The Predict Compositions
MS:MS_COMMENTS                   	node was set at 2 ppm mass tolerance with element counts C90 H190 N10 O18 P5 S5.
MS:MS_COMMENTS                   	A Compound Discoverer endogenous metabolites database with 4400 compounds was
MS:MS_COMMENTS                   	used to assign compound annotations with a mass tolerance of 5 ppm. Results
MS:MS_COMMENTS                   	obtained were further filtered by removing background from blank samples (75%
MS:MS_COMMENTS                   	ACN), removing features with no annotations from mass list, as well as manual
MS:MS_COMMENTS                   	curation.
MS:ION_MODE                      	NEGATIVE
MS:MS_RESULTS_FILE               	ST003119_AN005113_Results.txt	UNITS:Raw Area Count	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END