Microbially Mediated Anaerobic Carbon Cycling

In Limnologically Contrasting Perennially Ice-Covered Antarctic Lakes

This project is a collaborative effort between the Joye Research Group and Dr. Mike Madigan (Southern Illinois University Carbondale). We are working at sites in the Antarctic dry valleys, including Lake Fryxell and Lake Vanda. Researchers in the Joye group are quantifying rates of microbial processes, including anaerobic oxidation of methane, methanogenesis, and sulfate reduction; studying the controls on microbial metabolism; and using molecular ecological techniques to identify the microbes involved in key biogeochemical processes. We measure geochemical parameters in water and sediment samples, including concentrations of nutrients, redox species (e.g., sulfate, sulfide, reduced iron, dissolved inorganic carbon, etc.), organic matter (e.g., DOC, volatile fatty acids) and dissolved gases (e.g., CH4, N2O, H2) concentrations.

Anaerobic carbon cycling in Lakes Fryxell and Vanda

The decomposition (or mineralization) of organic matter recycles carbon and essential nutrients in terrestrial and aquatic environments. The basic processes involved in organic matter decomposition are fundamentally similar in all ecosystems, but variations in environmental factors can drive differences in mineralization rates and efficiencies (the amount of recycled organic matter relative to that which is deposited in sediments). Organic matter decomposition involves processes such as hydrolysis, fermentation, and terminal metabolism that are mediated by microorganisms. Organic matter mineralization rates are influenced by many factors, including organic matter reactivity, redox conditions and the availability of terminal electron acceptors, bulk ion concentration and composition, microbial community composition, and temperature. Bulk organic matter is a complex mixture of biomaterials with distinct reactivities; some materials (e.g. carbohydrates) degrade rapidly while others (e.g. lignin) are more resistant to degradation. The relative abundance of reactive versus recalcitrant compounds influences mineralization rates.

In Lakes Fryxell and Vanda, the bottom waters are anoxic, as are the sediments. These two lakes have different sulfate concentrations (lower in Fryxell than Vanda), which drive differences in both microbial community structure and activity. The presence or absence of sulfate determines to a large extent the relative production of CH4 versus CO2 during anaerobic degradation of organic carbon. In high sulfate sediments (e.g., marine sediments), sulfate is the primary oxidant and CO2 the primary product of organic carbon mineralization; methanogenesis becomes important in such habitats only after sulfate is depleted. In low sulfate sediments (e.g., freshwater sediments), methanogenesis is the dominant anaerobic terminal metabolic pathway and both CH4 and CO2 are produced. The key methanogens in most habitats are hydrogenotrophic methanogens, which convert CO2 + H2 to CH4, and acetoclastic methanogens, which split acetate to CH4 and CO2. In some cases, methanogenesis can be fueled by alternate substrates such as methylated amines, formate, or methanol. Homoacetogens, which convert H2 and CO2 to acetate, may play an important role in anaerobic carbon cycling as well, though this process is more poorly understood than sulfate reduction or methanogenesis. The role of microbial community composition on mineralization rates is less clear, but seasonal changes in microbial community structure may influence the rates and products of mineralization. Temperature influences mineralization rates directly by influencing microbial activity but may also affect distinct types of microorganisms to varying degrees. For example, homoacetogenesis appears to be favored over hydrogenotrophic methanogenesis at low temperature, so variable environmental temperatures can strongly regulate carbon flow under sulfate-depleted conditions.

Antarc_Fig1.jpg

A diverse microbial community mediates organic carbon mineralization in anaerobic habitats. Particulate organic matter is first hydrolyzed to high molecular weight dissolved organic matter (HMW-DOM) (left, figure 1). This HMW-DOM is further hydrolyzed and fermented to low molecular weight dissolved organic matter (LMW-DOM), which may undergo additional fermentation (primary fermentation) to generate alcohols, amino acids, and volatile fatty acids (VFA), including acetate. The products of primary fermentation may undergo secondary (2º) fermentation to smaller VFAs or they may be converted to hydrogen (H2), CO2, and CH4. Finally, the products of 2º fermentation are terminally metabolized to CH4 and/or CO2. Secondary fermentation reactions are critical in methanogenic habitats because methanogens require simple (C1, C2) substrates. Sulfate reducers usually out-compete methanogens for primary substrates (H2, acetate), so in sulfate-containing water or sediments, sulfate reduction rates usually exceed methane production rates by a significant margin. Competition for CO2 and H2 between hydrogenotrophic methanogens and acetogens may further regulate methane production in the environment.

Methane dynamics in anoxic habitats

A fascinating intersection of the sulfur and carbon cycles in anoxic habitats involves the anaerobic oxidation of methane (AOM). In stark contrast to other processes in the global CH4 cycle (e.g. methanogenesis; aerobic methanotrophy), AOM is somewhat of a microbiological and biogeochemical enigma. AOM occurs in the anoxic sediments and water columns of both freshwater and marine systems. However, the pathway(s) of AOM and the controls on AOM in the environment are unclear, and our understanding of the diversity and associations of microorganisms involved in AOM is incomplete. In marine sediments, measurements of AOM rates and modeling results suggest that up to 98% of the CH4 produced is oxidized anaerobically near the SO42-CH4 interface. Similarly, rates of AOM in the water column of hypersaline lakes effectively reduces (by up to 98%) the efflux of CH4 to the atmosphere.

The generally accepted biochemical mechanism of AOM in marine sediments involves syntrophic coupling between a CH4 oxidizer and a sulfate reducing bacterial (SRB) partner to effectively reverse the methanogenic CO2 reduction pathway (i.e. CH4 is oxidized to CO2 and electrons, released as either H2 or as an organic compound). Much of the available biomarker and molecular ecological data from marine systems supports the syntrophic consortium hypothesis and putative methanotrophic Archaea, and SRB partners have been identified in several environments. However, other molecular biological and biomarker data that suggest the involvement of multiple archaeal and bacterial groups in AOM, some of which are not associated with a syntrophic partner. Recent evidence documents the coupling of AOM and denitrification, so AOM may be supported by electron acceptors other than sulfate. It is likely that additional electron acceptors for AOM remain to be discovered. These data underscore the current poor understanding of the microbiological and biogeochemical conditions that support this globally critical process. 

Study Sites: Lakes Fryxell and Vanda

The McMurdo Dry Valleys (77°40’S, 163°E) are the largest nearly ice-free region on the Antarctic continent and are among the coldest and driest places on Earth.

Vanda_Ant3a.jpg

Nevertheless, life thrives in a variety of Dry Valley environments including cryptoendolithic communities within the interstitial spaces of rocks, soils, dry streambeds, ice, and in the water and sediments beneath the permanent lake ice. Life in these environments is limited to microorganisms and is sensitive to the balance between ice and liquid water. During cool summers, decreased stream flow and sublimation leads to loss of the lake ice cover, so lake volumes decrease and salinity increases. During mild summers, increased stream flow leads to increased lake volumes and reestablishment of ice covers. Repeated cycles of evaporation and refilling have characterized the McMurdo Dry Valley over time resulting in an unusual situation in the lakes found there: the lakes are geographically close but have dramatically different geochemistries with respect to salt content and solute composition.

The ice cover on the dry valley lakes eliminates wind-driven mixing, limiting vertical transport processes to molecular diffusion, and greatly limits gas exchange with the atmosphere, light penetration to the lake water, and sediment deposition to the lake bottom.

Fryxell_Ant2a.jpg

As a result, extreme biogeochemical disequilibria occurs. These systems have persistent stratification (amixis), reminiscent of meromictic (density-stratified) lakes. In meromictic lakes, strong gradients in bioactive constituents such as dissolved inorganic nitrogen exist; similar gradients are observed in these amictic lakes. During prolonged meromixis, waters trapped below the pycnocline (density gradient) become anoxic if there is no light to support oxygenic photosynthesis. Persistent anoxia in bottom water and sediments impacts the pathways and rates of organic carbon mineralization (Figure 1 above).

Although solutes in both Fryxell and Vanda originate from salt saturated brines that formed during the nearly complete evaporation of the lakes 1,000 to 3,000 years ago, the geochemistries of the two lakes are quite different. Fryxell contains brackish (0.73% w/v salt) anoxic, sulfidic bottom water containing ~1.5 mM sulfide with an overlying oxic freshwater layer; the solutes are primarily Na-Cl/HCO3-.

Fryxell_Ant2.jpg

There is clear evidence for sulfate reduction, methanogenesis, and anoxygenic photosynthesis in Fryxell bottom waters. Methanogenesis dominates the sediments of Fryxell.

Vanda, on the other hand, contains oxygenated fresh surface waters but anoxic, hypersaline water (10.85% w/v salt; ~ 4X seawater salinity) below 63 m. 

Vanda_Ant3.jpg

Vanda contains primarily a Ca-Cl brine, and the sulfate concentration is ~ 5 mM at the sediment water interface. Bottom water sulfate reduction mineralizes a large fraction of the total lake primary productivity in Vanda. We recently documented sulfate reduction in Vanda sediments but not in the anoxic water column. Interestingly, Vanda has an inverted temperature gradient, with a sub-ice temperature of < 4°C and a bottom water temperature of ~20°C. Fryxell contains high concentrations of DOC in bottom waters (up to 25 mg L-1; up to ~24% of the DOC is humic substances. Humic substances in Fryxell can serve as the electron acceptor for anaerobic acetate oxidation; we are exploring the role of humics in supporting AOM during this project. The bottom water and sediments of these two lakes thus experience very different thermal and biogeochemical environments.

Lake Sediments

The sediments of Antarctic lakes have received minimal study; to date there are only a few published studies using culture-independent approaches to examine the diversity of Antarctic lake sediments, and these studies were all of lakes outside the dry valleys. Our study is breaking important new ground. There has been no systematic study of the microbial diversity of dry valley lake bulk sediments using any methods. Methane and sulfur dynamics have been studied recently in two Antarctic lakes: Ace Lake and Lake Untersee. In both cases, methane was primarily produced in the sediments and consumed aerobically in the water column, a pattern typical of temperate freshwater lakes. By contrast, methane in Fryxell, produced in the anoxic sediments, is consumed in both the sediments and anoxic zones of the water column.

Results thus far

So far, our results are quite intriguing. We show that: (1) the rate of methanogenesis is ~30X higher in Fryxell bottom waters and sediments than in Vanda; (2) sulfate reduction occurs in both the water column and sediments of Fryxell but only in the sediments of Vanda; the sulfate reduction rate is ~14X higher in Vanda sediments than in Fryxell sediments; and, (3) AOM occurs in Fryxell in zones where sulfate is rapidly being consumed but highest AOM rates occur where sulfate concentrations are very low (Figure 2). Though methanogenesis in Fryxell occurs exclusively in the sediments, AOM occurs in both the upper layers of the sediments and in the water column (Figure 2).

Antarc_Fig4.jpg

Contrary to observations in other environments, rates of AOM in Fryxell bottom waters and sediments significantly exceed rates of sulfate reduction (see Table 1). Repeated rate measurements confirmed that the AOM rate exceeded the expected 1:1 stoichiometry with sulfate reduction (i.e. one CH4 oxidized to CO2 per SO42- reduced to H2S), indicating that another terminal electron acceptor is involved in AOM in Fryxell.

The disparity between AOM and sulfate reduction led us to conduct a series of lab experiments aimed at elucidating whether the traditional electron acceptors associated with AOM stimulated activity in Fryxell samples. We amended lake water with SO42-, NO3-, and tungstate (an inhibitor of SO42- reduction) and monitored the effect on AOM.

Ant_Table_1.jpg

AOM rates under in situ conditions were ~28 times SO42- reduction rates. While SO42- addition generated a moderate stimulation of sulfate reduction, AOM rates were not significantly higher (Table 1). Similarly, NO3- did not stimulate AOM. Tungstate completely inhibited SO42- reduction but had no effect on AOM, further showing that AOM and sulfate reduction are not coupled. These data provide strong evidence that some other electron acceptor supports AOM in Fryxell bottom waters and likely in sediments as well. We hypothesize that the electron acceptor may be humic derivatives or related organic compounds because of the high load of DOC and humics present in Fryxell. Available molecular evidence also points to novel prokaryotes involved in AOM in Fryxell. To date, no 16S rRNA gene sequences closely related to those linked to AOM in marine systems (where AOM is linked to sulfate reduction) or the novel organisms involved in methane oxidation coupled to nitrate have been observed in clone libraries or denaturing gradient gel electrophoresis (DGGE) fragments. Whether AOM occurs in Vanda remains to be determined; to date, we have not observed AOM activity in Vanda waters or sediments.

Research Questions

We are determining differences in biogeochemical signatures and quantifying rates and pathways of anaerobic carbon cycling in the bottom waters and sediments of Fryxell and Vanda. We are enriching and isolating microorganisms involved in carbon and sulfur cycling and characterizing the microbial community using culture-independent methods. Finally, we are conducting directed laboratory experiments to elucidate the mechanism of AOM in these unique habitats. This work is addressing the following overarching question: What factors control terminal carbon metabolism (sulfate reduction, methanogenesis, and methanotrophy) in anoxic and highly stable polar lake waters and sediments? What novel microbial communities are responsible for the AOM, and which electron acceptor(s) support AOM in these unique lakes? Work conducted to date has illustrated an abundance of novel microorganisms in the MCM Dry Valley lakes, and our preliminary work on sulfate reduction, methanogenesis and AOM points strongly to novel metabolic processes, including in particular the likelihood that previously unrecognized electron acceptors support AOM in Fryxell (Table 1). 

Our work is addressing the following questions:

  1. Do rates and/or mechanisms of anaerobic carbon and sulfur cycling differ between the anoxic bottom waters and sediments? If so, why?
  2. Is sulfate availability the main factor driving differences between anaerobic carbon/sulfur cycling in Fryxell and Vanda?
  3. Given the different thermal regimes, what, if any, role does temperature play in regulating microbial activity rates in these two lakes?
  4. What is the mechanism of AOM in the anoxic, sulfate-depleted bottom waters and sediments of Fryxell?
  5. Does AOM occur in the anoxic, sulfate-rich bottom waters and sediments of Vanda and if so, what is the mechanism?
  6. What novel microorganisms are involved in AOM and other methane- and sulfur-cycling processes in these lakes? How does microbial diversity determined from functional genes (mcrA, dsrAB) compare to that determined from 16S-rRNA gene analyses? 

Researchers

Vladimir Samarkin (Research Scientist)

Marshall Bowles (received his PhD 2011)

Charles Schutte (PhD student)

Melitza Crespo-Medina (was Postdoctoral Researcher 2010 - 2013)

Papers

Samarkin, V.A., M.T. Madigan, M.W. Bowles, K.L Casciotti, J. Priscu, C. McKay, and S.B. Joye, 2010. Abiotic brine-rock reactions produce nitrous oxide in a cold, hypersaline, Don Juan Pond, Antarctica. Nature Geoscience, 3: 341-344, doi:10.1028/ngeo847.

Dohm, J.M., H. Miyamoto, G.G. Ori, A.G. Fairen, A.F. Davila, G. Komatsu, W.C. Mahaney, P. Williams, S.B. Joye, and 27 others, 2010. Prime candidate environments for life detection on Mars. Geological Society of America, Frontiers Volume, “Analogs for Planetary Exploration”, Special Paper 483.

Presentations

Samarkin, V.A., M. Madigan, M. Bowles, and S.B.Joye. Novel mechanism of anaerobic methane oxidation in permanently ice-covered Lake Fryxell, Antarctica. Goldschmidt 2010 Conference, Knoxville, TN, June.

Kempher, M.L., G.S. Tregoning, S.B. Joye, and M.T. Madigan. Calcium-tolerant bacteria isolated from the deep waters of Lake Vanda, McMurdo Dry Valleys, Antarctica. Am. Soc. Microbiology, 110th General Meeting, San Diego, CA, May.

Funders

National Science Foundation Office of Polar Programs

NASA Exobiology program