Trace Metal Limitations on Methane Production in ...epsc.wustl.edu/~catalano/epsc595/Catalano_Metals_Methane_Wetland… · Overall emission rate determined by complex array of biotic

Trace Metal Limitations on Methane Production in Freshwater Wetland Soils

Washington University: Jeffrey G. Catalano, Nyssa M. Crompton, Alexander S. Bradley

Saint Louis University: Elizabeth A. HasenmuellerUniversity of Central Florida: Lisa G. Chambers

Acknowledgements

Earth and Planetary Sciences • Washington University

Andy Tappmeyer (MO Dept Conserv)Gary Calvert (MO Dept Conserv)UCF Arboretum StaffSam Webb (SSRL)Mike Pape (PNC/XSD, APS)Dale Brewe (PNC/XSD, APS)Sanmathi Chavalmane (WU – NRF)Jen Houghton (WU)

Financial SupportUS DOE Office of Biological & Environmental Research, Subsurface Biogeochemical Research Program

Scientific Support

Wetlands are a Major Source of the Greenhouse Gas Methane

■ Wetlands, primarily freshwater, are the largest natural source of CH4 emissions to the atmosphere– Wetland CH4 emissions are predicted to increase in a

warming climate■ CH4 is responsible for the second largest radiative

forcing of the well-mixed greenhouse gasesEarth and Planetary Sciences • Washington UniversityFrom: 2014 IPCC Report

Variability in T-Dependence of CH4 Emission Rates from Wetlands Indicates Metabolic Control

■ Aquatic environments display T-dependent CH4 emissions consistent with methanogen metabolisms (Ea = 1.10 eV/106 kJ mol-1) and different from respiration and photosynthesis

■ Overall emission rate determined by complex array of biotic and abiotic controls


General T-Dependence of CH4 Emissions

Yvon-Durocher et al. (2014) Nature 507, 488-491

0.96 eV93 kJ mol-1

Global Climate Models Include a Mechanistic Biogeochemical Model for Methane Emissions

■ Global models account for many local-scale controls on methane emissions beyond temperature and rainfall


Physical and Biogeochemical Processes Affecting CH4 Emissions

– Biogeochemical controls of CH4 production

– CH4 oxidation before release– Water saturation state

– Gas diffusion– Ebullition– Venting through aerenchyma

tissue in wetland plant roots

Predicted Average Annual CH4Emissions over a 25-Year Simulation

From: Riley et al. (2011) Biogeosciences

Trace Metal are Essential to Methanogen Metabolisms

■ All metabolic routes to methane production utilize a series of metalloenzymes– All pathways terminate through

methyl coenzyme M reductase, (mcr) which contains the Ni-bearing F430 cofactor

– Ni also used in hydrogenases– Co plays key roles in

methyltransferase enzymes– Zn involved in second to last

step (hdr) in methanogenesis– Mo or W needed for CO2 + H2

■ Methanogens require trace metals for growth– Optimal dissolved levels are

typically 1-5 μM, up to 100 μM for Fe

Earth and Planetary Sciences • Washington UniversityAfter: Glass and Orphan (2012) Front. Microbio.

Metalloenzymes in Methanogen Metabolic Pathways

Many Enzymes also Contain Iron

Trace Metal Limitations Demonstrated in Pure Cultures and Anaerobic Bioreactors

■ Importance of Ni, Co, and Mo for methanogens has been long established– Low trace metal availability limits

CH4 production in pure cultures and anaerobic digesters


Effect of [Ni] on the Growth Rate of Methanobacterium thermoautotrophicum

Schöenheit et al. (1979) Arch. Microbiol.

Control

Co+Mo

Ni+Mo

Ni+CoNi+Co+Mo

Enhanced Performance of Anaerobic BioreactorsMurray and van den Berg (1981) Appl. Env. Microbiol.

Control

Side Note: Trace Metal Availability is Assumed to Limit CH4 Production Through Earth History

■ Drop in the maximum Fe:Ni ratio in banded iron formations (BIFs) suggested to reflect a decline in marine Ni concentrations (~400 nM to ~100 nM)– Iron oxides proposed to have scavenged Ni from seawater– Decline in Ni availability hypothesized to have inhibited

methanogenesis around the time of the Great Oxidation Event (~2.5-2.3 Ga), allowing for the rise of oxygen


Ni Content of BIFs through Time

■ Trace metal limitation studies in field settings are rare– Generally not considered when

probing biogeochemistry■ One study of wetland peat soils

found metal additions to soil microcosms enhanced CH4production for 2 ombrotrophic sites– Peat soils from other sites showed

inhibition or no effect– Iron was added as Fe3+, a

competing electron acceptor that can also be used for CH4 oxidation

– Addition of other compounds had similar effects

■ It is thus unclear whether natural wetlands show trace metal limitations on CH4 production

Earth and Planetary Sciences • Washington UniversityBasiliko and Yavitt (2001) Biogeochem. 52, 133-153

Lack of Study of Metal Limitations in Natural Wetlands

CH

4

Potential Natural and Anthropogenic Effects on Methane Production via Trace Metals

■ Trace metal contents of soil and aquatic systems have a wide natural variation at a range of spatial scales

– These have been altered by historical and ongoing emissions and discharges from anthropogenic activities

– Wetlands also constructed to treat legacy metal contamination■ If they occur, trace metal limitations in wetland may vary substantially

across a region and be impacted by past and ongoing human activity


Ni in River Basin Stream SedimentsYager and Folger (2003) USGS MF-2407

Ni in U.S. Surface SoilsSmith et al. (2014) USGS OFR 2014-1082

Assessing Whether Natural Wetlands Display Trace Metal Limitations on CH4 Production

■ Our initial approach involves:– Field site characterization– Assessment of the controls on

metal availability

– Exploration of the effects of metal additions on CH4production in soil microcosms


Wetland Field Site Properties and Characterization


Missouri and Florida Field Sites


Both Sites are Peat-Based Wetlands but Have Different Vegetation and Hydrology

■ Missouri site MTC:– Stream-fed marsh with additional

groundwater inputs– Marsh grasses and Typha dominate– Soils contain a ~5 cm peat layer

overlying clay■ Florida site UCF:

– Depressional cypress dome swamp fed by precipitation and groundwater

– Soils consists of leaf litter overlying a 20 cm to 1 m thick muck and peat layer

■ Both sites are permanently saturated and free from anthropogenic water inputs (e.g., no industrial waters)


Field Characterization and Sampling

■ Sampling transects were established along hydrologic gradients■ Overlying surface waters were sampled for metals, nutrients, and

major elements■ Triplicate soil cores were collected

– Sealed in the field with O2 scavenger to limit oxygen exposure– Transferred to an anaerobic chamber in the laboratory for

characterization and experiments


Missouri Marsh Florida Swamp Soil Core from Missouri

Field Site Waters Differ in Composition but Both are Low

in Trace Metals

■ Missouri site has water composition of river water or groundwater, Florida site water is rain-derived with some groundwater inputs

■ Florida low in nutrients, close to O2 saturation■ Dissolved metal concentrations ~0.1 to 5% of

optimal levels for methanogenesis (1 to 5 μM)


Mo

BD

L

Mo

BD

L

Mo

BD

L

Dissolved Metals in Site Surface Waters

Site pH Ca (mg/L)

Na (mg/L)

Mg(mg/L)

K (mg/L)

Cl (mg/L)

MTC 6.6 216 23 36 8.6 20

UCF 6.6 1.9 4.4 0.66 0.35 8

Site DO(mg/L)

NH3(mg/L)

NO3(mg/L)

PT(mg/L)

ST(mg/L)

Fe (mg/L)

MTC 0.3 1.6 0.6 0.2 2.3 BDL

UCF 6.4 0.01 BDL 0.01 1.0 0.1

pH and Major Elements

Nutrients and Redox-Sensitive Species

Field Site Peat Soils Differ in Trace Metal, Iron, and Sulfur Contents

■ Florida site soil has substantially lower trace metal contents than observed for the Missouri site soil– Missouri soil contains ~100x the Fe and S content of the Florida soil– Sequential chemical extractions suggest that <1% of trace metals at

each site are likely available for solubilization and biological uptake■ The water and soil chemistry suggests that the Florida site has the

greater likelihood of displaying metal limitations, but both sites lack optimal levels of metals for methanogenesis


Site Ni (μg/g)

Co (μg/g)

Zn (μg/g)

Mo (μg/g)

Fe (wt.%)

S (wt.%) %OM

MTC 23 14 64 4.8 6.1 3.2 35.6

UCF 6.3 1.2 23 0.7 0.04 0.02 75.1

*Avg. Crust 47 17 67 1.1 3.9 0.06 -

Compositions of Upper Peat Layers at Field SitesSequential Extractions

*Rudnick and Gao (2012) Treatise Geochem.

Soil Microcosms and Metal Binding


Preliminary Soil Microcosms with Missouri Soil Showed No Effect of Metal Additions

■ The addition of individual metals or a mixture of trace metals produced no variation in CH4 production

■ Measurements of the final fluid composition revealed >99% binding of metals to the soil solids– The added metals did not actually increase metal availability


Addition Ni (μM)

Co (μM)

Zn (μM)

Mo (μM)

No metals 0.07 0.04 0.02 0.03

0.6 μmol Ni 0.07 0.05 0.01 0.03

0.3 μmol Co 0.06 0.06 0.01 0.04

0.3 μmol Zn 0.06 0.04 0.01 0.04

0.3 μmol Mo 0.05 0.04 0.01 1.60

Mixture 0.09 0.04 0.03 0.61

Final Dissolved Metal Concentrations

21±1°C, triplicate measurements, 3 g soil in 9 mL site water, N2 headspace, CH4 via GC-FID

0.01

0.1

1

10

100

1000

1 10 100 1000

Ni i

n so

lutio

n (µ

M)

Ni in Soil (µg/g soil)

0.01

0.1

1

10

100

1000

0.01 0.1 1 10 100

Ni i

n so

lutio

n (µ

M)

Ni Added (µmol/g soil)

0.01

0.1

1

10

100

1000

0.01 0.1 1 10 100

Ni i

n so

lutio

n (µ

M)

Ni Added (µmol/g soil)

■ Missouri peat soil shows high binding capacity for Ni– Saturating capacity and bringing Ni to optimal level requires 500

to 900 μg/g Ni in the solid phase■ Florida soil shows 10-50x lower binding capacity


0.01

0.1

1

10

100

1000

1 10 100 1000

Ni i

n so

lutio

n (µ

M)

Ni in Soil (µg/g soil)

Optimal for pure cultures

Optimal for pure cultures

Field Site Soils Show Distinct Nickel Binding Capacities

X-ray Absorption Spectroscopy Shows Field Site Soils Have Distinct Nickel Binding Mechanisms

■ XANES and EXAFS spectra show that added nickel binds through distinct mechanisms– Missouri: Binds to reduced sulfur, either thiol groups or a sulfide mineral– Florida: Binds to oxygen, likely carboxyl groups on soil OM

■ Recall: Missouri soil S content is 100x Florida soil (3.2 vs. 0.02 wt.%)


O SNi XANES Spectra Fourier Transform of Ni EXAFS Spectra

Sulfide Binding of Metals Known to Inhibit Methanogenesis in Anaerobic Bioreactors

■ Anaerobic bioreactors have been found to run optimally at ~2 μM dissolved Ni and Co– Ni and Co need to be added

when used to degrade waste or generate biogas

■ Substantially greater metal additions needed to actually optimize CH4 production

■ Sulfide in bioreactors binds Ni and Co

■ Initial soil microcosm behavior consistent with observations in bioreactors

Earth and Planetary Sciences • Washington UniversityGonzalez-Gill et al. (1999) AEM 65, 1789–1793

Added Co & Ni

Characterization of Sulfur and Metal Speciation in Wetland Soils


Speciation of the Low-Level Background Nickel is Similar at Both Sites

■ Nickel at both sites occurs as a mixture of sulfur-and carboxyl-bound species– Missouri: 35% to reduced S, 65% to carboxyl groups– Florida: 51% to reduced S, 49% to carboxyl groups


Iron-Sulfur-Organic Matter Associations in

Missouri Site Soil■ Strong Fe-S co-localization is

observed (R2 = 0.81)– Appear associated with likely

locations of OM– Discrete from Si, associated

with P but not Ca■ Portion of Ni is associated with

Fe-S regions


Calcium – Phorphorus – Silicon

Iron – Nickel – Sulfur500 μm

500 μm

Fe/Ca

Ni/PS/Si

Low Levels of Sulfur and Iron are Independent in

Florida Site Soil■ S and Fe are poorly correlated

(R2 = 0.15)– S is associated with OM

aggregates– Ca is associated with S, OM– Minor detrital Si

■ Ni is undetectable except for one possible Ni-S grain


Calcium – Phorphorus – Silicon

Iron – Nickel – Sulfur

500 μm

500 μm

Fe/Ca

Ni/PS/Si

Sulfur Micro-XANES Spectra Reveal Distinct S Chemistry at

the Two Sites■ Sulfur speciation in the

Missouri peat soil shows much greater variability than in the Florida soil– Principal component analysis

(PCA)* reveals 8 distinct sulfur components in Missouri, 3 in Florida

■ Clear signature of iron sulfide minerals in Missouri soil

■ Stronger signature of sulfonate, organosulfate, thiol groups in Florida

■ Inorganic SO4 is undetectable


FeS

FeS 2

, S0 ,

R-S

-S-R

R-S

H, c

yclic

-S

R-S

O3

R-O

-SO

3, Su

lfate

Mis

sour

iFl

orid

a

*Subset of spectra shown here: 38 total for MTC, 19 for UCF

Spectral Fitting Support Complex Sulfur Chemistry, Observes Sulfide Minerals Only at

the Missouri Site

■ Large microscale variability in S speciation in Missouri soil– Sulfide minerals, primarily FeS,

are widespread• Elemental S also occurs

– Reduced organic sulfur species are abundant

– Some intermediate to oxidized organic sulfur also occurs

■ Florida soil shows lesser variability– Mixture of organic sulfur

species

Earth and Planetary Sciences • Washington University • 28

Sulfide Minerals and Organic Sulfur Show

Overlapping Microscale Spatial Distributions

■ PCA-derived sulfur species maps show widespread organic sulfur in Missouri soil

■ Iron sulfide minerals have discrete occurrences but often overlap organic S

■ Iron map shows correlation with both iron sulfides and organic sulfur– Portion of Fe is associated with

organic matter■ High metal binding capacity in

Missouri likely derives from both mineral and organic sulfur


500 μm

Optimizing Soil Microcosms to Evaluate Metal Limitations


■ 50% increase in CH4 production over 2 weeks when binding capacity is exceeded, providing increased available nickel

■ Ni concentration still below optimal levels for pure cultures


Addition Ni (μM)

Co (μM)

Zn (μM)

Mo (μM)

No metals 0.14 0.02 0.11 0.16

30 μmol Ni 0.23 0.01 0.26 0.05


Exceeding Nickel Binding Capacity Enhance CH4 Production from Missouri Soil

21±1°C, triplicate measurements, 0.5 g soil in 1.5 mL site water, N2 headspace, CH4 via GC-FID

Greater Metal Additions Inhibited CH4Production

■ Drop in CH4 production correlated with very high residual dissolved metal concentrations– High Ni addition caused release of other metals from soil


Addition Ni (μM)

Co (μM)

Zn (μM)

Mo (μM)

No metals 0.14 0.02 0.11 0.16

30 μmol Ni 0.23 0.01 0.26 0.05

150 μmol Ni 6900 0.27 0.44 0.43

Mixture 1100 1600 880 3600


Dec

reas

e w

ith

Hig

h M

etal

s


High Metal Concentrations are Toxic to Methanogens

■ Studies of pure cultures show that elevated metals (mM levels) are toxic to methanogens

■ Elevated sulfide also inhibits methanogenesis through either direct toxicity or metal binding


Metal Toxicity Effects on Pure Cultures of MethanogensAfter: Sanchez et al. (1996) Lett. Appl. Microbio. 23, 439-444

Control

Optimized Ni Additions to Missouri Microcosms Generate up to 10x More CH4 than Controls

■ Adding nickel at levels that exceed the soil binding capacity generates a substantial enhancement in CH4 production– Co and Zn concentrations remain low but apparently not inhibitory

■ Nickel addition enhances Fe availability, presumably by competitive binding to reduced sulfur


Addition Ni (μM) Co (μM) Zn (μM) Fe (μM)

No metals 0.1 0.04 0.04 BDL

15 μmol Ni 1.1 0.01 BDL 3.1

30 μmol Ni 0.5 0.01 0.1 56

45 μmol Ni 1.0 0.01 0.04 147

60 μmol Ni 4.8 BDL BDL 280



10x

Incr

ease

Optimized Metal Additions to Florida Microcosms Yield No Enhancement in CH4 Production

■ Addition of nickel or mixtures of trace metals and iron bring concentrations to optimal levels

■ Lack of increase in CH4 production indicates that the Florida site soil is not limited by metal availability– Also shows that ample CH4 production can occur at low metal levels

Earth and Planetary Sciences • Washington University21±1°C, triplicate measurements, 1.5 g soil in 4.5 mL site water, N2 headspace, CH4 via GC-FID

Addition Ni (μM) Co (μM) Zn (μM) Fe (μM)

No metals 0.10 0.01 0.21 2.9

0.25 μmol Ni 0.65 BDL 0.05 3.1

0.5 μmol Ni 1.1 BDL 0.09 3.7

1.0 μmol Ni 2.1 0.01 0.16 9.4

Mixture 0.58 0.45 0.33 5.3


Summary and Implications


Summary of Major Findings

■ Wetland sites contain dissolved metals at concentration far below optimal for methanogenesis based on predictions for pure cultures

■ Soils at the Missouri wetland have a larger background pool of metals but also a large metal binding capacity compared to Florida soils– Difference correlates with sulfur content and presence of sulfide minerals and

reduced organic sulfur groups■ Ni addition enhances CH4 production is the Missouri site soil by up to

10x once the metal binding capacity is saturated■ Florida soils show a negligible response when adequate trace metals

are provided, but produce CH4 even when apparently metal-limited


Uncertain Nature of Metal Limitations on Methanogenesis in the Environment

■ Our systems suggest that dissolved metal concentrations are poor predictors of adequate availability– Integrating porewater samplers, such as

diffusive gels, may better indicate field availability of metals

■ Predictions from pure culture may not be transferable to the field– Methanogenesis may proceed at lower

metal availability than expected■ Sulfur has a key role in controlling the

occurrence of metal limitations– May be more prevalent in marine and

estuarine systems and under early Earth conditions that were sulfidic

■ Need to also understand archaeal community as acetoclastic and hydrogenotrophic methanogens may have different metal requirements


Sulfur Plays Multiple Key Roles in Controlling Methane Emissions from Freshwater Wetlands

■ Despite typically low abundance in freshwater wetlands, sulfate was recently shown to drive substantial anaerobic methane oxidation in such systems, limiting emissions

■ Our work shows that freshwater wetlands with ample sulfur contents may also limit emissions via inhibited production

■ Role of sulfur in freshwater wetlands is likely underestimated


CH4 Oxidation Coupled to Sulfate Reduction in Freshwater Wetlands

Segarra et al. (2015) Nature Comm.

Soil Sulfur Induces Trace Metal Limitations in Freshwater Wetlands

Major Outstanding Questions

■ What happens in wetlands with intermediate sulfur contents?

■ What is the relative importance of organic and inorganic sulfur in controlling metal availability?

■ Why do S-poor systems behave differently from pure cultures? Are methanogen communities in natural systems better optimized for metal acquisition?

■ Does trace metal availability limit other biogeochemical processes?– N2O reduction (Cu) and Hg

methylation (Co) have high potential for metal limitation

Earth and Planetary Sciences • Washington UniversityImages from: Pester et al. (2012) Front. Micro.; Macomber & Hausinger(2011) Metallomics; Creative Commons

Rates of S Cycling in Freshwater Wetlands Have Been Underestimated

High- and Low-Affinity Ni Transporters

Wide Array of Essential Metalloenzymes


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Trace Metal Limitations on Methane Production in ...epsc.wustl.edu/~catalano/epsc595/Catalano_Metals_Methane_Wetland… · Overall emission rate determined by complex array of biotic