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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
Wetland Field Site Properties and Characterization
Earth and Planetary Sciences • Washington University
Missouri and Florida Field Sites
Earth and Planetary Sciences • Washington University
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)
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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)
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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.%)
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
500 μm
Optimizing Soil Microcosms to Evaluate Metal Limitations
Earth and Planetary Sciences • Washington University
■ 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
Earth and Planetary Sciences • Washington University
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
Final Dissolved Metal Concentrations
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
Earth and Planetary Sciences • Washington University
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
Final Dissolved Metal Concentrations
Dec
reas
e w
ith
Hig
h M
etal
s
21±1°C, triplicate measurements, 0.5 g soil in 1.5 mL site water, N2 headspace, CH4 via GC-FID
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Final Dissolved Metal Concentrations
21±1°C, triplicate measurements, 0.5 g soil in 1.5 mL site water, N2 headspace, CH4 via GC-FID
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
Final Dissolved Metal Concentrations
Summary and Implications
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University
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
Earth and Planetary Sciences • Washington University