Microbial Communities Driving Carbon Sequestration in Coastal Estuarine Wetlands

Leah Van Dyke¹, Ty Samo², Buck Hanson³, Rhona Stuart², Aaron Chew², Mike Allen², Jeff Kimbrell² Jennifer Pett-Ridge² and Adina Paytan¹

¹University of California, Santa Cruz ; ²Lawrence-Livermore National Laboratory; ³Los Alamos National Laboratory

Wu et al. 2023

Wetlands are hotspots for carbon sequestration.

• Coastal wetlands compose 2% of the total ocean cover and store 50% of the carbon buried in the ocean (NOAA)

Microorganisms play an important role in chemical exchanges between soils and the atmosphere

• In order to understand how and when wetlands operate as a carbon sink or a carbon source, we must study the underlying microbial processes within them and how microbial taxa react to seasonal and annual environmental changes.

What are the impacts of wetland restoration…

• on microbial community structure and function?
• who the active microbes are across different wetland sites?
• on microbial populations cycling carbon, specifically CH₄?

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Research goals:

• quantify microbial biomass using microscopy
• assess differential microbial composition
• identify active microbes (¹⁸O-SIP)
• quantify CH₄ cycling genes (DNA) and expression (RNA) using qPCR targeting genetic markers (mcrA, pmoA, mmoX)

“Pristine” : Yampah

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A marsh that has not been altered, but is at risk for loss due to sea level rise. It has muddy soil, abundant algae, and higher plant cover than the “restored” site.

“Restored” : Hester

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Sandy soil was added to raise the elevation of the marsh to avoid flooding due to sea-level rise. The sandy soil was compacted during restoration. Plant cover is lower than at the pristine site, but is increasing.

Eddy Flux

Pristine

Restored

Graphic made by Sylvain Labedens In the Paytan Lab

Sample Curves

Relative abundance of DNA in ng/uL is plotted by isotope: ¹⁶O control seawater, and ¹⁸O seawater

• ESP: Pristine Site “Yampah” ; ESR: Restored Site “Hester”

Incorporation of ¹⁸O into DNA will cause a shift to the right (heavier density fractions)

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Top 25 “enriched” ASVs per site.

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There is no overlap between the top 25 most active ASVs, as identified by EAF values, in the Pristine and Restored Sites.

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The top 25 most active at the “Pristine” are more “enriched” than Top 25 for the “Restored” site.

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Let’s look at the top 4 in each site…

EAF stands for Excess Atom Fraction, which is the proportion of labeled oxygen atoms (¹⁸O) that are assimilated into the DNA of an organism actively growing and synthesizing DNA.

• An EAF value of 0.2 means 20% of the oxygen atoms within the 16S gene are ¹⁸O labeled.

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ASV710

ASV946

ASV55

ASV1942

ASV944

ASV229

ASV127

ASV1179

Top 4 Pristine

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Top 4 Pristine

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Top 4 Pristine

Top 4 Restored

2 SOB (ASV127 and ASV 944), and Aquipluma is an anaerobic bacteria capable of nitrate reduction

All Aerobic bacteria typical in soil, marine sediments, and/or saline environments.

Restored Plots

Pristine Plots

1. Pristine: Clearer shift towards higher density gradients (more enriched in ¹⁸O)

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1. Restored: Less obvious of a shift towards higher density gradients (less enriched in ¹⁸O)

Summary

The “Pristine” Site and the “Restored” site have differences in the composition of most-active taxa and activity of those taxa.

• The top 25 most-active taxa are different at each site.
• The top 25 taxa in the “Restored” site are less active than the top 25 most active taxa in the “Pristine” site as tracked by EAF calculations using qSIP

Next Steps: I will use the taxonomies, and compare overall composition and active taxa composition across sites.

• Are the most active taxa also the most abundant?
• If I combine similar taxa by genus or class, how does this impact my interpretations of relative activity?
• How does this change in the wet season? (2024 samples)

Differences in Microbial Biomass Between Pristine and Restored Sites Observed Using Microscopy 2024 Wet Season

→ Blue dots = bacteria stained with DAPI

→ Larger red structures = chloroplast autofluorescence

2024 Wet Season Samples

Restored

Pristine

Top 5 cm

Top 5 cm

Microbial Cell Size Response to Light/Dark Incubation

Binary selection of individual heterotrophs

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Binary selection individual chloroplasts

Heterotrophs were largest in the Restored dark and Pristine light incubations. Cell size is an indicator of health and nutrient availability

‘24 Wet Season

Pristine

Heterotroph and Chloroplast Biomass Relative Abundance

Differences in response to dark/light incubations may be related to the relative abundance of chloroplast biomass.

‘24 Wet Season

‘24 Wet Season

Pristine

Summary

• Restored versus intact sites in Elkhorn Slough appear visually distinct at the microscale.
• Heterotrophs were largest in the Restored dark and Pristine light incubations
• There was lower relative abundance of chloroplast biomass in the Restored site than the Pristine Site

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• Yampah has been shown to fix more Carbon, but abundant heterotrophs may also metabolize more Carbon to CO₂ and CH₄ .
• 2024 wet season qSIP will tell us more about algal biomass across sites and response to light/dark incubations since we are adding 18S rRNA sequencing (eukaryote)

qPCR for Gene Copy Quantification at Depth

K Chico & J Piczon 2023

What are the impacts of wetland restoration…

−on microbial populations cycling carbon, specifically CH₄?

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-quantify CH₄ cycling genes (DNA) and expression (RNA) using qPCR targeting genetic markers (mcrA, pmoA, mmoX)

Dissimilatory Sulfite Reductase

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Dissimilatory sulfite reductase (DSR) is an enzyme in the final step of SO₄²⁻ reduction, and converts SO₃²⁻ to S²⁻.

The dsrB gene encodes the beta subunit of DSR.

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SRB plays a key role in anaerobic organic matter degradation, using SO₄²⁻ as an electron acceptor, producing sulfide as a byproduct.

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A large amount of H₂S production from sulfate reducers can compete with methanogens for H⁺.

Kushkevych et. al 2021

3 Representative Sites

North

Hester

Yampah

Pristine

Restored

Flooded

A marsh that has not been altered, but is at risk for loss due to sea level rise. It has muddy soil, abundant algae, and higher plant cover than the “restored” site.

Sandy soil was added to raise the elevation of the marsh to avoid flooding due to sea-level rise, and was compacted during restoration. Plant cover is lower than at the pristine site, but is increasing.

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The site had been previously diked and drained for farming, which compacted the soils. It currently experiences limited tidal exchange, and is a mudflat.

Eddy Flux

Pristine

Restored

Flooded

Graphic made by Sylvain Labedens In the Paytan Lab

Bring empty, pre-labeled tubes

Flash freeze sample in the field in lq. N₂

or if lq N2 is not available, put on dry ice in the field,

or put on ice in the field, flash freeze when possible

Store @ -80C

~1/8^th or 1-2 g (or whatever fits in a 2 ml tube)

Bring pre-labeled tubes containing 0.5 ml of 4% formaldehyde

Aliquot a small amount of sediment (~0.1 g or a cap full)

within 24 h, centrifuge (4C) to pellet.

remove sup., resuspend in 1 ml 1X PBS.

Centrifuge to pellet

remove sup.

resuspend in 0.5 ml 50% ethanol:1XPBS.

Store @ -20C

Used for nucleic

acid analyses

Used for microscopic

imaging

Yampah

3 replicate cores

Hester & North

~10 sections per core (or whatever seems reasonable in the field), section depth should be consistent or noted

qPCR

qPCR (quantitative Polymerase Chain Reaction) amplifies DNA to quantify specific genetic material in a sample.

• Fluorescent markers bind to the DNA, and a machine measures the fluorescence after each cycle, indicating the DNA quantity.
• By tracking fluorescence across cycles, we can estimate the initial DNA concentration.

Keer 2008

Hamamatsu

The standard curve establishes the relationship between known DNA concentrations and their fluorescence.

The SQ (starting quantity) value is calculated by comparing the sample's fluorescence to a standard curve, indicating the initial DNA concentration.

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X- axis: cycle #, y-axis: RFU (flourescence)

X- axis: log SQ, y-axis: Cq

R^2: 0.945

Open circles: Standards

X’s: unknown samples

dsrB is more abundant than the other two genes.

• The high H₂S gas measured indicates strong sulfate reduction activity.

• Methanogenic genes are lowest at the Yampa. They are highest at the other 2 sites which are CH₄ sources.
• Methanotrophic gene, pmoA, was detected at all sites but was typically lower than methanogenic gene, mcrA, levels.

Summary

pmoA, mcrA, and dsrB were detected at all three representative sites.

• dsrB was more abundant than the other two genes.
• Methanogenic genes were present across all sites but were lowest at the Pristine site (Yampah) and highest at the other 2 sites which are CH₄ sources.
• Methanotrophic gene, pmoA, was detected at all sites but was typically lower than methanogenic gene, mcrA, levels.

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Strong sulfate reduction activity may outcompete methanogens for H⁺, limiting CH₄ flux at some sites. Additional data for mmoX, a methanotrophic gene, are not yet measured, and may alter our understanding of these dynamics.

Quantify CH₄ cycling genes (DNA) using qPCR

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Quantify microbial biomass using microscopy

Assess differential microbial composition & Identify active microbes

1. Heterotrophs were largest in the Restored dark and Pristine light incubations
2. There was lower relative abundance of chloroplast biomass in the Restored site.

1. The top 25 most-active taxa are different at each site.
2. The top 25 taxa in the “Restored” site are less active than in the “Pristine” site.

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1. dsrB, mcrA, and pmoA genes were detected at all sites, with dsrB being the most abundant.
2. Methanogenic genes were highest at CH₄ source sites. Methanotrophic genes were consistently lower, than methanogenic genes.

Questions?