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 con....

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-i.......

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