Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Summary

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

Abstract

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

Introduction

Coastal wetlands are vital ecosystems that contribute to climate regulation, biodiversity conservation, and water management through processes such as carbon sequestration, evapotranspiration, and methane (CH₄) emissions¹. These ecosystems, including both freshwater and saltwater wetlands², are highly productive and act as critical zones for uptake of carbon dioxide (CO₂) and capture organic matter from terrestrial and marine environments^3,4. The dynamic interactions within these wetlands stimulate microbial CH₄ production and consumption⁵, positioning them as one of the largest natural sources of CH₄⁶. As the second most important greenhouse gas, CH₄ has a global warming potential approximately 27-30x greater than that of CO₂^4,7,8,9, making the study of CH₄ emissions from coastal wetlands essential in the era of climate change. The emission of CH₄ is influenced by various environmental factors, particularly salinity, playing a crucial role in microbial processes¹⁰. Freshwater wetlands contribute significantly to atmospheric methane due to their lower sulfate levels, which facilitates greater microbial CH₄ production, whereas saltwater wetlands generally tend to emit less CH₄ due to higher sulfate concentrations^11,12,13.

CH₄ emissions from coastal wetlands are generally controlled by two groups of microorganisms, known as methanogens and methanotrophs¹⁴. Methanogens produce CH₄ in anoxic sediments by breaking down substrates like formate, acetate, hydrogen, or methylated compounds through a process known as methanogenesis¹⁵. The important enzyme in this pathway is methyl-coenzyme M reductase (MCR), as it catalyzes the final and rate-limiting step of methanogenesis^15,16,17. The mcrA gene, which encodes the alpha subunit of MCR, is a functional marker that can be found in all methanogenic archaea¹⁸. Moreover, in coastal wetlands, the sulfate-methane transition zone (SMTZ) forms above the methanogenic zone, where methane diffusing upward and sulfate moving downward converge and are depleted¹⁹. Within this zone, anaerobic methanotrophic archaea (ANME) oxidize methane to carbon dioxide using the MCR enzyme, while sulfate-reducing bacteria (SRB) reduce sulfate to sulfide. SRB outcompete methanogens for hydrogen and acetate, limiting methane production until sulfate is depleted^16,17.

In contrast, aerobic methanotrophic bacteria oxidize CH₄ in aerobic environments²⁰, utilizing different forms of methane monooxygenase (MMO). These include particulate methane monooxygenase (pMMO), a copper-containing enzyme embedded in the intracytoplasmic membrane, and soluble methane monooxygenase (sMMO), an iron-containing enzyme found in the cytoplasm. However, for pMMO, there are three gene operons pmoCAB²¹; among them, pmoA gene is the most conservative for all the methanotrophs. There are two different biomarker genes for pmoA: pmoA1 and pmoA2²². Moreover, for a comprehensive understanding of methanotrophs, mmoX gene is used as a tool in molecular biology to identify sMMO-containing methanotrophs²³. This distinction in metabolic pathways and environmental requirements of methanogens and aerobic methanotrophs highlights the complex microbial interactions regulating methane cycling in coastal wetland ecosystems.

The Boca Chica (BC) wetland, a productive saltwater environment in South Texas, experiences tidal influences from the Gulf of Mexico (GOM), leading to variable surface salinity levels, especially due to its proximity to the hypersaline Laguna Madre²⁴. This tidal action, alternating between high and low tides, causes oxygen levels to fluctuate²⁵ that might alter methanogen and methanotroph activity in sediments²⁶. In contrast, coastal freshwater wetlands are considered to be a significant hotspot for CH₄ fluxes²⁷. The coastal freshwater wetlands in South Texas, including Resaca Del Rancho Viejo (RV) and Lozano Banco (LB), distant from the GOM's tidal effects, have distinct hydrological management. RV experiences pulse flows supplemented by river water during low water levels, whereas LB operates as an offline flow system without such supplementation. Moreover, RV and LB maintain lower salinity levels due to a high discharge of artificially pumped freshwater and being an oxbow lake, respectively. The different environmental factors can significantly influence methane cycling across South Texas coastal wetlands. However, methane cycling in South Texas coastal wetlands remains an area that has yet to be thoroughly investigated.

Polymerase chain reaction (PCR) and real-time PCR (also called quantitative PCR [qPCR]) represent fundamental and widely utilized techniques for detecting and quantifying the relative abundance of specific genes in environmental samples. These techniques specifically amplify targeted regions of DNA to indicate the presence and relative quantity of CH₄ cycling-related genes, providing indicators of potential methane cycling. Nevertheless, the availability and efficacy of PCR primer sets might be limited by various inhibitory factors in the extracted environmental DNA, being impacted by the types of environments^28,29. Thus, this study mainly established an optimal PCR method for detecting the presence of CH₄ cycling-related genes in South Texas coastal wetlands (Figure 1) and then visualized their quantified relative abundance in these ecosystems. The results from this study can be applied to other coastal regions to enhance the understanding of CH₄ cycling and microbial dynamics in diverse coastal ecosystems.

Protocol

1. Sample collection

1. Collect sediment samples using a sediment grab sampler or shovel.
   NOTE: Samples were collected from two stations of three distinct coastal wetlands during cool (October-February, the average temperature is 20 °C) and warm (April-June, average temperature is 27 °C) seasons of 2023 and 2024. A sediment grab sampler was used when samples were collected from coastal freshwater wetlands (Figure 2) and a shovel was used for tidal-influenced coastal saltwater wetlands.
2. Lower the sampler into the shallow waterbody first and allow it to sink to the sediment surface (within the top 50 cm) under its own weight, minimizing disturbance to the sediment structure as shown in Figure 2.
3. Pull it out of the water column where the depth was typically 60 cm to 215 cm³⁰, transfer the sediment samples to zip-lock bags, and store them in an ice box immediately.
   NOTE: Clean and wash the grab sampler with deionized (DI) water before proceeding to the next stations.
4. Store all samples at -20 °C immediately in the laboratory.
5. In each sampling site, measure surface water quality parameters such as salinity and temperature in situ using a multiparameter water quality meter.
   NOTE: The probes were rinsed with deionized water (DI water) after use in each station.

2. Genomic DNA extraction

1. Thaw the samples at room temperature before starting the procedure for genomic DNA extraction.
2. Transfer approximately 500 mg of sediment samples to a 15 mL tube and centrifuge at 4,250 × g for 3 min to remove all water.
3. Extract genomic DNA using a DNA extraction kit for soil following the manufacturer's protocol³¹ with a little modification and store at -20 °C immediately.
   NOTE: The changes were made to reduce redundancy and efficient workflow.
   1. Add up to 500 mg of soil sample to a glass bead/ceramic sphere-containing tube.
   2. Add 978 µL of Sodium Phosphate Buffer to the sample in the bead/sphere-containing tube.
   3. Add 122 µL of buffer lysis solution to the sample in the bead/sphere-containing tube to solubilize external contaminants.
   4. Homogenize using a bead mill homogenizer at 5x the speed level for 20 s and repeat 2x.
      NOTE: A bead mill homogenizer was used in this study, which is why the speed was adjusted.
   5. Centrifuge the mixture for 10 min at 14,000 × g.
   6. Transfer the supernatant to a clean 2.0 mL microcentrifuge tube.
   7. Add 250 µL of protein precipitation solution (PPS) to separate the solubilized nucleic acids from the cellular debris and lysing matrix. Mix by inverting the tube 10x.
   8. Centrifuge at 14,000 × g for 5 min to precipitate the pellet, removing the cellular debris and lysing matrix.
   9. Transfer the supernatant to a clean 15 mL microcentrifuge tube.
   10. Add 1.0 mL of the Binding Matrix suspension to the supernatant in the 15 mL tube.
       NOTE: Shake the Binding Matrix suspension to resuspend before adding it.
   11. Allow the binding of DNA to the Binding Matrix by placing the tubes on a rotator for 2 min.
   12. Place all the tubes on a rack and incubate for 3 min to allow the settling of the Binding Matrix.
   13. After 3 min, discard 750 µL and gently mix the remaining supernatant with the pellet using a pipette.
   14. Transfer 750 µL of the mixture to a SPIN filter and centrifuge at 14,000 × g for 1 min. Empty the catch tube and reuse it. Repeat with the remaining mixture.
   15. Add 500 µL of the prepared wash solution (with the appropriate amount of ethanol added) to further solubilize impurities. Gently resuspend the pellet using the force of the liquid from the pipet tip.
   16. Centrifuge at 14,000 × g for 1 min to remove impurities. Empty the catch tube and reuse it.
   17. Centrifuge again at 14,000 × g for 2 min without adding anything.
   18. Replace the catch tube with a new, clean catch tube and air dry the SPIN Filter for 5 min at room temperature.
   19. Add 50 µL DNAse free water (DES) and centrifuge at 14,000 × g for 1 min.
       NOTE: In this protocol, 50 µL of DES was used for DNA elution to ensure optimal recovery and stability of the extracted environmental DNA (eDNA).

3. DNA quantification

1. Add 1 µL of extracted DNA with 200 µL of fluorescent dye in a 0.5 mL tube and mix thoroughly by pipetting.
2. Wrap the tube immediately with aluminum foil so light cannot penetrate and incubate at room temperature for 5 min.
3. Measure DNA concentration by using a fluorometer at ONE DNA concentration mode following the manufacturer's protocol.

4. Detection of 16S rRNA, pmoA1 , pmoA2 , mmoX , and mcrA by conventional PCR

1. Before running conventional PCR (cPCR), thaw all samples and reagents in an ice bucket.
2. Dilute all extracted eDNA samples to 10 ng/µL.
   NOTE: A list of primers is given in Table 1.
3. Prepare a 25 µL cPCR reaction mixture for each sample, including 12.5 µL of 2x PCR Master mix , 0.5 µL of forward and reverse primers (10 µM) (see Table 1 for primer list), 1 µL of 10 ng/µL eDNA, and 10.5 µL of nuclease-free water.
   NOTE: Prepare the master mix with enough volume for one extra sample to minimize pipetting errors.
4. Perform cPCR reaction following the protocol consisting of an initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 45 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 m, with varying annealing temperature for different primers (see Table 1 for annealing temperatures of different primers used for different genes).
   NOTE: For mcrA gene, ML primers showed the desired band using a slow ramp rate of 0.1 °C/s between the annealing and extension steps for the first five cycles³².
5. Visualize cPCR products in an agarose gel prestained with ethidium bromide (EtBr).
   NOTE: Use 2.5% agarose gel for a 50 bp ladder and 0.9% gel for a 1 kb ladder. Use 1x TAE buffer for a 2.5% agarose gel and 0.5x TAE buffer for a 0.9% agarose gel.

5. Detection of pmoA1 , pmoA2 , mmoX, and mcrA by quantitative real-time PCR

NOTE: Methanogen- and methanotroph-targeted genes such as pmoA1, pmoA2, mmoX, and mcrA abundance were observed by qPCR using a real-Time PCR system.

1. Prepare the standards for each gene separately to obtain the standard curve for each gene.
   1. Amplify the targeted gene with cPCR, using the samples that produced the brightest band during the gel electrophoresis of each gene. Follow the method and primers described in section 4.
   2. Purify the amplified products using a gel extraction kit and measure the DNA concentration following the method described in section 3 and store at -20 °C.
      NOTE: Aliquot the purified standards into separate tubes for further use.
   3. Calculate gene copies from the measured DNA concentration using the copy number calculating website for qPCR.
   4. Dilute the calculated copy number of the standard with nuclease-free water to prepare each standard, ranging from 10⁸ to 10² copies/µL, before running the qPCR.
   5. Prepare the standard curve using three replicates of each copy number, including a negative control (NTC, no template DNA).
      NOTE: The R² value of each standard curve was greater than 0.99.
2. Prepare a 20 µL qPCR reaction mixture for all samples, standards, and NTC. Perform the qPCR analysis in triplicates for all samples.
   1. Place all reaction components, including qPCR master mix, primers, nuclease-free water, standards, and samples, on an ice rack before beginning.
   2. Prepare a 20 µL qPCR reaction mixture for each sample, standard, and NTC, containing 10 µL of SYBR Green master mix, 0.5 µL of each 10 µM forward and reverse primers, 8 µL of nuclease-free water, and 1 µL of either 10 ng/µL template DNA, or standard, or DI water respectively.
      NOTE: Use the primer sets shown to yield optimal results for pmoA1 and mcrA genes in conventional PCR for qPCR (see Table 1). To improve accuracy, run each sample in triplicate. The following steps provide an efficient method for preparing triplicate samples with a combined volume of 60 µL per sample.
      1. Prepare the reaction mixture to combine the master mix, forward and reverse primers for the specific gene, and nuclease-free water in a 2 mL tube, except the template DNA.
         NOTE: Prepare the reaction mixture volume considering pipetting error. For example, if there are 24 samples and 8 standards, calculate the total volume for 33 reactions instead of 32 reactions to minimize pipetting errors. In this case, the total volume required for triplicate reactions would be as follows: 990 µL of master mix (33 samples x 3 replicates x 10 µL), 49.5 µL of forward primers (33 samples x 3 replicates x 0.5 µL), 49.5 µL of reverse primers (33 samples x 3 replicates x 0.5 µL), and 792 µL of nuclease-free water (33 samples x 3 replicates x 8 µL).
      2. Prepare PCR tubes according to the number of samples and standards.
      3. Dispense 57 µL of the prepared reaction mixture into each PCR tube.
         NOTE: To perform qPCR for each sample in triplicate, prepare a total reaction mixture volume of 57 µL per sample (excluding template DNA, standard, or water). This volume will be divided equally into three wells for one sample, with 19 µL allocated to each well.
      4. Add 3 µL of template DNA, standard, or nuclease-free water to each tube and mix by gently tapping the tube bottom.
         NOTE: The total volume of reaction mixture prepared for one sample would be 60 µL now in each tube.
   3. Aliquot 20 µL of the prepared reaction mixture from each tube into the designated wells of a 96-well qPCR plate. Seal the PCR plate with adhesive PCR Sealing Film using an applicator.
   4. Centrifuge the sealed plate at 1,000 × g for 1 min to ensure proper mixing to eliminate any bubbles within the wells.
3. Place the PCR plate in the thermal cycler. Turn on the qPCR machine and then open the related software to set up the protocol.
   1. Set up the protocol according to the qPCR master mix guidelines. Use the following protocol: 95 °C for 10 min, followed by 95 °C for 15 s, and an extension step at 72 °C for 30 s. Perform the annealing step at the annealing temperature specified for the relevant primers in Table 1 for 45 s. Conduct all qPCR runs for 35 cycles.
   2. Set up the 96-well qPCR plate with standards and NTCs in the same configuration as the sample-containing plate.
4. Use the absolute quantification standard curve method to quantify the amplified product and the gene copy number in each sample³³.

6. Visualizing methane-cycling genes in the map of South Texas Coastal wetlands

1. Open geographic information system (GIS) software ArcGIS Pro and save the project file with the name Study Area in the specified folder on the computer.
2. Click on Map in the top left | Basemap and select Terrain with Labels as the basemap.
3. Click on Locate | Search and when the search bar opens up, locate the study area by typing the area's name; the area will show up.
4. Draw the specific area using georeferencing.
   1. Click on View | Catalog Pane from the top layer.
   2. Double-click on Folder from the catalog | File Name.
   3. Right-click on geodatabase (.gdb) file and then click on New | Feature Class and Create Feature Class will show up.
   4. Type Name and Alias box and click Finish at the bottom.
   5. Click on View | Contents. The Alias name will show up in the Contents Pane.
   6. Click on Edit from the top layer | Create. The Create Features pane will open. Double-click on the Alias in Create Features pane, and the Configure Tool Feedback Options will appear.
   7. Select Lines, then sketch lines on the map to create an outside boundary of the study area. Double-click on the map when finished.
5. Minimize GIS software, then open a spreadsheet. Type the sample name in the first column, enter the latitude in the second column, and the longitude in the third column. Use the next four columns for the qPCR data of pmoA1, pmoA2, mmoX, and mcrA.
6. Save the file in CSV format in any specific folder of the computer.
7. Open the GIS software again and click Add Data | XY Point Data.
8. Select the CSV file from the folder on the computer in the Input Table box. Rename the file name in the Output Feature Class, then click Run to display the sampling points on the map.
9. Click on thevsearch bar at the top and search Kriging.
10. Select the sampling station file and then select pmoA1.
11. Click the Environment | select the sampling station in layer and mask | click Run.
12. Follow the protocols mentioned in steps 6.9, 6.10, and 6.11 to create Kriging for pmoA2, mmoX, and mcrA for all the study area.
13. Create a layout of the map.
    1. Click on Insert from the top layer | New Layout, and select ANSI - Landscape.
    2. Click on Map Frame, select the map with the Kriging, and place all the maps in the layout by drawing a rectangle. This will make the map visible in the layout.
    3. Select the North Arrow and place it in the layout to indicate the North direction.
    4. Select Scale Bar to display the scale of the area on the map.
    5. Click on Legend to display the legends, then place it in the layout.
    6. Click Grid and select any of the black graticule options. This will create the grid with latitude and longitude and display it in the Contents Pane with the label Black Horizontal Label Graticule.
    7. Double-click on Black Horizontal Label Graticule, then select Components. Click on Ticks 1 and Grid, and remove these components by clicking on the cross sign to their right.
14. Click on Share from the top layer, then click Export Layout. Select the file type as PDF, save the file on the computer using the Name Box, set the vertical resolution to 500 DPI, and click Export to create the PDF file of the map.

Results

To understand the distribution and abundance of CH₄ cycling-related genes (mcrA, pmoA1, pmoA2, and mmoX) in the coastal wetlands of South Texas, the extracted eDNA from each sample was analyzed by cPCR and qPCR. Universal primers for each biomarker were selected to run cPCR from previous studies (Table 1)^22,34,35,36,

Discussion

Coastal wetlands are recognized as significant contributors to atmospheric methane, an important greenhouse gas⁴⁰. Although there have been studies on methane flux and methanogens in wetlands^41,42,43, little is known about how methanotrophs operate across different environments or under various management practices, especially in wetlands with fluctuating water levels⁴⁴. Moreover, ...

Materials

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	ThermoFisher Scientific	AB0620	https://www.thermofisher.com/order/catalog/product/AB0620?SID=srch-srp-AB0620
0.5 mL PCR Tubes	Promega	E4941	https://www.promega.com/products/biochemicals-and-labware/tips-and-accessories/0_5ml-pcr-tubes/?catNum=E4941
10 μL tips	ThermoFisher Scientific	05-408-187	Fisherbrand SureGrip Pipet Tip Racked or Reload System Tips Natural; 10μL; | Fisher Scientific
15 mL centrifuge tube	ThermoFisher Scientific	14-959-53A	https://www.fishersci.com/shop/products/falcon-15ml-conical-centrifuge-tubes-5/p-193301
200 μL tips	ThermoFisher Scientific	05-408-190	Fisherbrand SureGrip Pipet Tip Racked or Reload System Tips Natural; 200μL; | Fisher Scientific
1000 μL tips	ThermoFisher Scientific	02-707-402	https://www.fishersci.com/shop/products/sureone-micropoint-pipette-tips-specific-standard-fit/02707402?gclid=Cj0KCQiAp NW6BhD5ARIsACmEb kUsQ9Lu0YIq5i4vWege 17qPdtxIYZyvmJH1cDo ARuwereO1V4GLz9UaA lDREALw_wcB&ef_id=C j0KCQiApNW6BhD5ARI sACmEbkUsQ9Lu0YIq5i 4vWege17qPdtxIYZyvmJ H1cDoARuwereO1V4GLz 9UaAlDREALw_wcB:G:s &ppc_id=PLA_goog_2175 7693617_171052169911_02 707402__715434303113_1555 377385658230343&ev_chn=sh op&s_kwcid=AL!4428!3!71543430 3113!!!g!2366517300713!&gad_source=1
Applied Biosystem Power SYBR Green Master Mix	ThermoFisher Scientific	4368577	https://www.thermofisher.com/order/catalog/product/4368577
ArcGIS Pro	esri		https://www.esri.com/en-us/arcgis/products/arcgis-pro/overview?srsltid=AfmBOopatJ4 JvHJfscHRcAaDx0Jz5_Jrl8l5 vYkkBvfOqE-uNSsMghN1
CFX Duet Real-Time PCR system	Bio-Rad	12016265	https://www.bio-rad.com/en-us/product/cfx-duet-real-time-pcr-system?ID=97722926-9ed9-16a4-1d83-c92f587e427a
Corning Lambda plus single channel pipettor volume 0.5-10 μL	Sigma-Aldrich	CLS4071-1EA	https://www.sigmaaldrich.com/US/en/product/sigma/cls4071
Corning Lambda plus single channel pipettor volume 100-1000 μL	Sigma-Aldrich	CLS4075-1EA	https://www.sigmaaldrich.com/US/en/product/sigma/cls4075
Corning Lambda plus single channel pipettor volume 20-200 μL	Sigma-Aldrich	CLS4074-1EA	https://www.sigmaaldrich.com/US/en/product/sigma/cls4074
FastDNA spin kit for soil	MP Biomedical	116560200-CF	https://www.mpbio.com/us/116560000-fastdna-spin-kit-for-soil-samp-cf?srsltid=AfmBOoqOxxGilzY3IHNIZR ajegGTr9MoX1oMZUh 3dcbJqe0UvvukY128
Gene copy  calculator	Science Primer		https://scienceprimer.com/copy-number-calculator-for-realtime-pcr .
High speed benchtop centrifuge	ThermoFisher Scientific	75004241	https://newlifescientific.com/products/thermo-scientific-sorvall-st16-high-speed-benchtop-centrifuge-75004241?gad_source=1&gclid=Cj0KCQiApN W6BhD5ARIsACmEbkVC_-cCIN9j 20TvYq8iDsBlUR5cPK_1_wN OBEcjMdv-CYVoGCfeOLYaAv enEALw_wcB
High speed microcentrifuge	VWR	75838-336	https://us.vwr.com/store/product/20546590/null
Lysing Matrix E tube			glass bead/ceramic sphere-containing tube
Microcentrifuge tube	ThermoFisher Scientific	02-681-320	https://www.fishersci.com/shop/products/fisherbrand-low-retention-microcentrifuge-tubes-8/02681320?gclid=Cj0KCQiAp NW6BhD5ARIsACm EbkWbG4_o3oUiGk HJPU-_31-CuexDwQ fmWPnfyhBOf2BHXsy K3fFW1toaAgJbEALw_ wcB&ef_id=Cj0KCQiAp NW6BhD5ARIsACmEb kWbG4_o3oUiGkHJPU- _31-CuexDwQfmWPnfy hBOf2BHXsyK3fFW1toa AgJbEALw_wcB:G:s&ppc _id=PLA_goog_21757693 617_171052169911_0268 1320__715434303113_10 349826094968484711&ev _chn=shop&s_kwcid=AL!4 428!3!715434303113!!!g!23 66517300713!&gad_source=1
PCR Master mix	Promega	M7502	https://www.promega.com/products/pcr/taq-polymerase/master-mix-pcr/?catNum=M7502
Quantiflour ONE dsDNA system	Promega	E4871	https://www.promega.com/products/rna-analysis/dna-and-rna-quantitation/quantifluor-one-dsdna-system/?gad_source=1&gbraid=0AAAAAD _rg189yJTY3cxeVqMdu8RPx10 Ma&gclid=CjwKCAjwxNW2BhAk EiwA24Cm9FUgViPNyWq7UfZL VeeoroLAZ5JIP6w07RGK_4D0w oZgAqf-G1XTmxoCxm8QAvD_B wE&catNum=E4871
Quantus Fluorometer	Promega	E6150	https://www.promega.com/products/microplate-readers-fluorometers-luminometers/fluorometers/quantus-fluorometer/?catNum=E6150
YSI Pro 2030	YSI a xylem brand	603174	https://www.ysi.com/product/id-p2030/pro2030-kits