Resolving the influence of lignin on soil organic matter decomposition with mechanistic models and continental-scale data.

Confidence in model estimates of soil CO2 flux depends on assumptions regarding fundamental mechanisms that control the decomposition of litter and soil organic carbon (SOC). Multiple hypotheses have been proposed to explain the role of lignin, an abundant and complex biopolymer that may limit decomposition. We tested competing mechanisms using data-model fusion with modified versions of the CN-SIM model and a 571-day laboratory incubation dataset where decomposition of litter, lignin, and SOC was measured across 80 soil samples from the National Ecological Observatory Network. We found that lignin decomposition consistently decreased over time in 65 samples, whereas in the other 15 samples, lignin decomposition subsequently increased. These "lagged-peak" samples can be predicted by low soil pH, high extractable Mn, and fungal community composition as measured by ITS PC2 (the second principal component of an ordination of fungal ITS amplicon sequences). The highest-performing model incorporated soil biogeochemical factors and daily dynamics of substrate availability (labile bulk litter:lignin) that jointly represented two hypotheses (C substrate limitation and co-metabolism) previously thought to influence lignin decomposition. In contrast, models representing either hypothesis alone were biased and underestimated cumulative decomposition. Our findings reconcile competing hypotheses of lignin decomposition and suggest the need to precisely represent the role of lignin and consider soil metal and fungal characteristics to accurately estimate decomposition in Earth-system models.

Contrasting geochemical and fungal controls on decomposition of lignin and soil carbon at continental scale.

Lignin is an abundant and complex plant polymer that may limit litter decomposition, yet lignin is sometimes a minor constituent of soil organic carbon (SOC). Accounting for diversity in soil characteristics might reconcile this apparent contradiction. Tracking decomposition of a lignin/litter mixture and SOC across different North American mineral soils using lab and field incubations, here we show that cumulative lignin decomposition varies 18-fold among soils and is strongly correlated with bulk litter decomposition, but not SOC decomposition. Climate legacy predicts decomposition in the lab, and impacts of nitrogen availability are minor compared with geochemical and microbial properties. Lignin decomposition increases with some metals and fungal taxa, whereas SOC decomposition decreases with metals and is weakly related with fungi. Decoupling of lignin and SOC decomposition and their contrasting biogeochemical drivers indicate that lignin is not necessarily a bottleneck for SOC decomposition and can explain variable contributions of lignin to SOC among ecosystems.

Extreme low-flow conditions in a dual-chamber denitrification bioreactor contribute to pollution swapping with low landscape-scale impact.

Denitrification bioreactors are an effective edge-of-field conservation practice for nitrate (NO3) reduction from subsurface drainage. However, these systems may produce other pollutants and greenhouse gases during NO3 removal. Here a dual-chamber woodchip bioreactor system experiencing extreme low-flow conditions was monitored for its spatiotemporal NO3 and total organic carbon dynamics in the drainage water. Near complete removal of NO3 was observed in both bioreactor chambers in the first two years of monitoring (2019-2020) and in the third year of monitoring in chamber A, with significant (p < 0.01) reduction of the NO3-N each year in both chambers with 8.6-11.4 mg NO3-N L-1 removed on average. Based on the NO3 removal observed, spatial monitoring of sulfate (SO4), dissolved methane (CH4), and dissolved nitrous oxide (N2O) gases was added in the third year of monitoring (2021). In 2021, chambers A and B had median hydraulic residence times (HRTs) of 64 h and 39 h, respectively, due to varying elevations of the chambers, with drought conditions making the differences more pronounced. In 2021, significant production of dissolved CH4 was observed at rates of 0.54 g CH4-C m-3 d-1 and 0.07 g CH4-C m-3 d-1 in chambers A and B, respectively. In chamber A, significant removal (p < 0.01) of SO4 (0.23 g SO4 m-3 d-1) and dissolved N2O (0.21 mg N2O-N m-2 d-1) were observed, whereas chamber B produced N2O (0.36 mg N2O-N m-2 d-1). Considering the carbon dioxide equivalents (CO2e) on an annual basis, chamber A had loads (~12,000 kg CO2e ha-1 y-1) greater than comparable poorly drained agricultural soils; however, the landscape-scale impact was small (<1 % change in CO2e) when expressed over the drainage area treated by the bioreactor. Under low-flow conditions, pollution swapping in woodchip bioreactors can be reduced at HRTs <50 h and NO3 concentrations >2 mg N L-1.

Poorly drained depressions can be hotspots of nutrient leaching from agricultural soils.

Much of the US Corn Belt has been drained with subsurface tile to improve crop production, yet poorly drained depressions often still flood intermittently, suppressing crop growth. Impacts of depressions on field-scale nutrient leaching are unclear. Poor drainage might promote denitrification and physicochemical retention of phosphorus (P), but ample availability of water and nutrients might exacerbate nutrient leaching from cropped depressions. We monitored nitrate, ammonium, and reactive P leaching across multiple depression-to-upland transects in north-central Iowa, using resin lysimeters buried and retrieved on an annual basis. Crops included conventional corn/soybean (Zea mays/Glycine max) rotations measured at fields with and without a winter rye (Secale cereale) cover crop, as well as juvenile miscanthus (Miscanthus × giganteus), a perennial grass. Leaching of nitrogen (N) and P was greater in depressions than in uplands for most transects and years. The median difference in nutrient leaching between paired depressions and uplands was 56 kg N ha-1 year-1 for nitrate (p = 0.0008), 0.6 kg N ha-1 year-1 for ammonium (p = 0.03), and 2.4 kg P ha-1 year-1 for reactive P (p = 0.006). Transects managed with a cover crop or miscanthus tended to have a smaller median difference in nitrate (but not ammonium or P) leaching between depressions and uplands. Cropped depressions may be disproportionate sources of N and P to downstream waters despite their generally poor drainage characteristics, and targeted management with cover crops or perennials might partially mitigate these impacts for N, but not necessarily for P.

Shared Microbial Taxa Respond Predictably to Cyclic Time-Varying Oxygen Limitation in Two Disparate Soils.

Periodic oxygen (O2) limitation in humid terrestrial soils likely influences microbial composition, but whether communities share similar responses in disparate environments remains unclear. To test if specific microbial taxa share consistent responses to anoxia in radically different soils, we incubated a rainforest Oxisol and cropland Mollisol under cyclic, time-varying anoxic/oxic cycles in the laboratory. Both soils are known to experience anoxic periods of days to weeks under field conditions; our incubation treatments consisted of anoxic periods of 0, 2, 4, 8, or 12 d followed by 4 d of oxic conditions, repeated for a total of 384 d. Taxa measured by 16S rRNA gene sequences after 48 d and 384 d of experimental treatments varied strongly with increasing anoxic period duration, and responses to anoxia often differed between soils at multiple taxonomic levels. Only 19% of the 30,356 operational taxonomic units (OTUs) occurred in both soils, and most OTUs did not respond consistently to O2 treatments. However, the OTUs present in both soils were disproportionally abundant, comprising 50% of sequences, and they often had a similar response to anoxic period duration in both soils (p < 0.0001). Overall, 67 OTUs, 36 families, 15 orders, 10 classes, and two phyla had significant and directionally consistent (positive or negative) responses to anoxic period duration in both soils. Prominent OTUs and taxonomic groups increasing with anoxic period duration in both soils included actinomycetes (Micromonosporaceae), numerous Ruminococcaceae, possible metal reducers (Anaeromyxobacter) or oxidizers (Candidatus Koribacter), methanogens (Methanomicrobia), and methanotrophs (Methylocystaceae). OTUs decreasing with anoxic duration in both soils included nitrifiers (Nitrospira) and ubiquitous unidentified Bradyrhizobiaceae and Micromonosporaceae. Even within the same genus, different OTUs occasionally showed strong positive or negative responses to anoxic duration (e.g., Dactylosporangium in the Actinobacteria), highlighting a potential for adaptation or niche partitioning in variable-O2 environments. Overall, brief anoxic periods impacted the abundance of certain microbial taxa in predictable ways, suggesting that microbial community data may partially reflect and integrate spatiotemporal differences in O2 availability within and among soils.

Temperature-dependence of metabolism and fuel selection from cells to whole organisms.

Temperature affects nearly every aspect of how organisms interact with and are constrained by their environment. Measures of organismal energetics, such as metabolic rate, are highly temperature-dependent and governed through temperature effects on rates of biochemical reactions. Characterizing the relationships among levels of biological organization can lend insight into how temperature affects whole-organism function. We tested the temperature dependence of cellular oxygen consumption and its relationship to whole-animal metabolic rate in garter snakes (Thamnophis elegans). Additionally, we tested whether thermal responses were linked to shifts in the fuel source oxidized to support metabolism with the use of carbon stable isotopes. Our results demonstrate temperature dependence of metabolic rates across levels of biological organization. Cellular (basal, adenosine triphosphate-linked) and whole-animal rates of respiration increased with temperature but were not correlated within or among individuals, suggesting that variation in whole-animal metabolic rates is not due simply to variation at the cellular level, but rather other interacting factors across scales of biological organization. Counter to trends observed during fasting, elevated temperature did not alter fuel selection (i.e., natural-abundance stable carbon isotope composition in breath, δ13 Cbreath ). This consistency suggests the maintenance and oxidation of a single fuel source supporting metabolism across a broad range of metabolic demands.

Nitrous oxide emissions from agricultural soils challenge climate sustainability in the US Corn Belt.

Agricultural landscapes are the largest source of anthropogenic nitrous oxide (N2O) emissions, but their specific sources and magnitudes remain contested. In the US Corn Belt, a globally important N2O source, in-field soil emissions were reportedly too small to account for N2O measured in the regional atmosphere, and disproportionately high N2O emissions from intermittent streams have been invoked to explain the discrepancy. We collected 3 y of high-frequency (4-h) measurements across a topographic gradient, including a very poorly drained (intermittently flooded) depression and adjacent upland soils. Mean annual N2O emissions from this corn-soybean rotation (7.8 kg of N2O-N ha-1⋅y-1) were similar to a previous regional top-down estimate, regardless of landscape position. Synthesizing other Corn Belt studies, we found mean emissions of 5.6 kg of N2O-N ha-1⋅y-1 from soils with similar drainage to our transect (moderately well-drained to very poorly drained), which collectively comprise 60% of corn-soybean-cultivated soils. In contrast, strictly well-drained soils averaged only 2.3 kg of N2O-N ha-1⋅y-1 Our results imply that in-field N2O emissions from soils with moderately to severely impaired drainage are similar to regional mean values and that N2O emissions from well-drained soils are not representative of the broader Corn Belt. On the basis of carbon dioxide equivalents, the warming effect of direct N2O emissions from our transect was twofold greater than optimistic soil carbon gains achievable from agricultural practice changes. Despite the recent focus on soil carbon sequestration, addressing N2O emissions from wet Corn Belt soils may have greater leverage in achieving climate sustainability.

MetaFunPrimer: an Environment-Specific, High-Throughput Primer Design Tool for Improved Quantification of Target Genes.

Genes belonging to the same functional group may include numerous and variable gene sequences, making characterizing and quantifying difficult. Therefore, high-throughput design tools are needed to simultaneously create primers for improved quantification of target genes. We developed MetaFunPrimer, a bioinformatic pipeline, to design primers for numerous genes of interest. This tool also enables gene target prioritization based on ranking the presence of genes in user-defined references, such as environment-specific metagenomes. Given inputs of protein and nucleotide sequences for gene targets of interest and an accompanying set of reference metagenomes or genomes, MetaFunPrimer generates primers for ranked genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 publicly available soil metagenomes. We demonstrate computationally that these amoA-AOB primers can cover 94% of the amoA-AOB genes observed in the 1,550 soil metagenomes compared with a 49% estimated coverage by previously published primers. Finally, we verified the utility of these primer sets in incubation experiments that used long-term nitrogen fertilized or unfertilized soils. High-throughput quantitative PCR (qPCR) results and statistical analyses showed significant differences in relative quantification patterns between the two soils, and subsequent absolute quantifications also confirmed that target genes enumerated by six selected primer pairs were significantly more abundant in the nitrogen-fertilized soils. This new tool gives microbial ecologists a new approach to assess functional gene abundance and related microbial community dynamics quickly and affordably. IMPORTANCE Amplification-based gene characterization allows for sensitive and specific quantification of functional genes. There is often a large diversity of genes represented for functional gene groups, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high-throughput primer design for various genes of interest and also allows for ranking gene targets by their presence and abundance in environmental data sets. Primers designed by this tool improve the characterization and quantification of functional genes in broad gene amplification platforms and can be powerful with high-throughput qPCR approaches.

High carbon losses from oxygen-limited soils challenge biogeochemical theory and model assumptions.

Oxygen (O2 ) limitation contributes to persistence of large carbon (C) stocks in saturated soils. However, many soils experience spatiotemporal O2  fluctuations impacted by climate and land-use change, and O2 -mediated climate feedbacks from soil greenhouse gas emissions remain poorly constrained. Current theory and models posit that anoxia uniformly suppresses carbon (C) decomposition. Here we show that periodic anoxia may sustain or even stimulate decomposition over weeks to months in two disparate soils by increasing turnover and/or size of fast-cycling C pools relative to static oxic conditions, and by sustaining decomposition of reduced organic molecules. Cumulative C losses did not decrease consistently as cumulative O2 exposure decreased. After >1 year, soils anoxic for 75% of the time had similar C losses as the oxic control but nearly threefold greater climate impact on a CO2 -equivalent basis (20-year timescale) due to high methane (CH4 ) emission. A mechanistic model incorporating current theory closely reproduced oxic control results but systematically underestimated C losses under O2  fluctuations. Using a model-experiment integration (ModEx) approach, we found that models were improved by varying microbial maintenance respiration and the fraction of CH4 production in total C mineralization as a function of O2 availability. Consistent with thermodynamic expectations, the calibrated models predicted lower microbial C-use efficiency with increasing anoxic duration in one soil; in the other soil, dynamic organo-mineral interactions implied by our empirical data but not represented in the model may have obscured this relationship. In both soils, the updated model was better able to capture transient spikes in C mineralization that occurred following anoxic-oxic transitions, where decomposition from the fluctuating-O2 treatments greatly exceeded the control. Overall, our data-model comparison indicates that incorporating emergent biogeochemical properties of soil O2 variability will be critical for effectively modeling C-climate feedbacks in humid ecosystems.

From pools to flow: The PROMISE framework for new insights on soil carbon cycling in a changing world.

Soils represent the largest terrestrial reservoir of organic carbon, and the balance between soil organic carbon (SOC) formation and loss will drive powerful carbon-climate feedbacks over the coming century. To date, efforts to predict SOC dynamics have rested on pool-based models, which assume classes of SOC with internally homogenous physicochemical properties. However, emerging evidence suggests that soil carbon turnover is not dominantly controlled by the chemistry of carbon inputs, but rather by restrictions on microbial access to organic matter in the spatially heterogeneous soil environment. The dynamic processes that control the physicochemical protection of carbon translate poorly to pool-based SOC models; as a result, we are challenged to mechanistically predict how environmental change will impact movement of carbon between soils and the atmosphere. Here, we propose a novel conceptual framework to explore controls on belowground carbon cycling: Probabilistic Representation of Organic Matter Interactions within the Soil Environment (PROMISE). In contrast to traditional model frameworks, PROMISE does not attempt to define carbon pools united by common thermodynamic or functional attributes. Rather, the PROMISE concept considers how SOC cycling rates are governed by the stochastic processes that influence the proximity between microbial decomposers and organic matter, with emphasis on their physical location in the soil matrix. We illustrate the applications of this framework with a new biogeochemical simulation model that traces the fate of individual carbon atoms as they interact with their environment, undergoing biochemical transformations and moving through the soil pore space. We also discuss how the PROMISE framework reshapes dialogue around issues related to SOC management in a changing world. We intend the PROMISE framework to spur the development of new hypotheses, analytical tools, and model structures across disciplines that will illuminate mechanistic controls on the flow of carbon between plant, soil, and atmospheric pools.

Conventional analysis methods underestimate the plant-available pools of calcium, magnesium and potassium in forest soils.

The plant-available pools of calcium, magnesium and potassium are assumed to be stored in the soil as exchangeable cations adsorbed on the cation exchange complex. In numerous forest ecosystems, despite very low plant-available pools, elevated forest productivities are sustained. We hypothesize that trees access nutrient sources in the soil that are currently unaccounted by conventional soil analysis methods. We carried out an isotopic dilution assay to quantify the plant-available pools of calcium, magnesium and potassium and trace the soil phases that support these pools in 143 individual soil samples covering 3 climatic zones and 5 different soil types. For 81%, 87% and 90% of the soil samples (respectively for Ca, Mg and K), the plant-available pools measured by isotopic dilution were greater than the conventional exchangeable pool. This additional pool is most likely supported by secondary non-crystalline mineral phases in interaction with soil organic matter and represents in many cases (respectively 43%, 27% and 47% of the soil samples) a substantial amount of plant-available nutrient cations (50% greater than the conventional exchangeable pools) that is likely to play an essential role in the biogeochemical functioning of forest ecosystems, in particular when the resources of Ca, Mg and K are low.

Lignin lags, leads, or limits the decomposition of litter and soil organic carbon.

Lignin's role in litter and soil organic carbon (SOC) decomposition remains contentious. Lignin decomposition was traditionally thought to increase during midstage litter decomposition, when cellulose occlusion by lignin began to limit mass loss. Alternatively, lignin decomposition could be greatest in fresh litter as a consequence of co-metabolism, and lignin might decompose faster than bulk SOC. To test these competing hypotheses, we incubated 10 forest soils with C4 grass litter (amended with 13 C-labeled or unlabeled lignin) over 2 yr and measured soil respiration and its isotope composition. Early lignin decomposition was greatest in 5 of 10 soils, consistent with the co-metabolism hypothesis. However, lignin decomposition peaked 6-24 months later in the other five soils, consistent with the substrate-limitation hypothesis; these soils were highly acidic. Rates of lignin, litter, and SOC decomposition tended to converge over time. Cumulative lignin decomposition was never greater than SOC decomposition; lignin decomposition was significantly lower than SOC decomposition in six soils. Net nitrogen mineralization predicted lignin decomposition ratios relative to litter and SOC. Although the onset of lignin decomposition can indeed be rapid, lignin still presents a likely bottleneck in litter and SOC decomposition, meriting a reconsideration of lignin's role in modern decomposition paradigms.

Interactive global change factors mitigate soil aggregation and carbon change in a semi-arid grassland.

The ongoing global change is multi-faceted, but the interactive effects of multiple drivers on the persistence of soil carbon (C) are poorly understood. We examined the effects of warming, reactive nitrogen (N) inputs (12 g N m-2  year-1 ) and altered precipitation (+ or - 30% ambient) on soil aggregates and mineral-associated C in a 4 year manipulation experiment with a semi-arid grassland on China's Loess Plateau. Our results showed that in the absence of N inputs, precipitation additions significantly enhanced soil aggregation and promoted the coupling between aggregation and both soil fungal biomass and exchangeable Mg2+ . However, N inputs negated the promotional effects of increased precipitation, mainly through suppressing fungal growth and altering soil pH and clay-Mg2+ -OC bridging. Warming increased C content in the mineral-associated fraction, likely by increasing inputs of root-derived C, and reducing turnover of existing mineral-associated C due to suppression of fungal growth and soil respiration. Together, our results provide new insights into the potential mechanisms through which multiple global change factors control soil C persistence in arid and semi-arid grasslands. These findings suggest that the interactive effects among global change factors should be incorporated to predict the soil C dynamics under future global change scenarios.

Iron-mediated organic matter decomposition in humid soils can counteract protection.

Soil organic matter (SOM) is correlated with reactive iron (Fe) in humid soils, but Fe also promotes SOM decomposition when oxygen (O2) becomes limited. Here we quantify Fe-mediated OM protection vs. decomposition by adding 13C dissolved organic matter (DOM) and 57FeII to soil slurries incubated under static or fluctuating O2. We find Fe uniformly protects OM only under static oxic conditions, and only when Fe and DOM are added together: de novo reactive FeIII phases suppress DOM and SOM mineralization by 35 and 47%, respectively. Conversely, adding 57FeII alone increases SOM mineralization by 8% following oxidation to 57FeIII. Under O2 limitation, de novo reactive 57FeIII phases are preferentially reduced, increasing anaerobic mineralization of DOM and SOM by 74% and 32‒41%, respectively. Periodic O2 limitation is common in humid soils, so Fe does not intrinsically protect OM; rather reactive Fe phases require their own physiochemical protection to contribute to OM persistence.

Trade-offs in soil carbon protection mechanisms under aerobic and anaerobic conditions.

Oxygen (O2 ) limitation is generally understood to suppress oil carbon (C) decomposition and is a key mechanism impacting terrestrial C stocks under global change. Yet, O2 limitation may differentially impact kinetic or thermodynamic versus physicochemical C protection mechanisms, challenging our understanding of how soil C may respond to climate-mediated changes in O2 dynamics. Although O2 limitation may suppress decomposition of new litter C inputs, release of physicochemically protected C due to iron (Fe) reduction could potentially sustain soil C losses. To test this trade-off, we incubated two disparate upland soils that experience periodic O2 limitation-a tropical rainforest Oxisol and a temperate cropland Mollisol-with added litter under either aerobic (control) or anaerobic conditions for 1 year. Anoxia suppressed total C loss by 27% in the Oxisol and by 41% in the Mollisol relative to the control, mainly due to the decrease in litter-C decomposition. However, anoxia sustained or even increased decomposition of native soil-C (11.0% vs. 12.4% in the control for the Oxisol and 12.5% vs. 5.3% in the control for the Mollisol, in terms of initial soil C mass), and it stimulated losses of metal- or mineral-associated C. Solid-state 13 C nuclear magnetic resonance spectroscopy demonstrated that anaerobic conditions decreased protein-derived C but increased lignin- and carbohydrate-C relative to the control. Our results indicate a trade-off between physicochemical and kinetic/thermodynamic C protection mechanisms under anaerobic conditions, whereby decreased decomposition of litter C was compensated by more extensive loss of mineral-associated soil C in both soils. This challenges the common assumption that anoxia inherently protects soil C and illustrates the vulnerability of mineral-associated C under anaerobic events characteristic of a warmer and wetter future climate.

Lower soil carbon stocks in exotic vs. native grasslands are driven by carbonate losses.

Global change includes invasion by exotic (nonnative) plant species and altered precipitation patterns, and these factors may affect terrestrial carbon (C) storage. We measured soil C changes in experimental mixtures of all exotic or all native grassland plant species under two levels of summer drought stress (0 and +128 mm). After 8 yr, soils were sampled in 10-cm increments to 100-cm depth to determine if soil C differed among treatments in deeper soils. Total soil C (organic + inorganic) content was significantly higher under native than exotic plantings, and differences increased with depth. Surprisingly, differences after 8 yr in C were due to carbonate and not organic C fractions, where carbonate was ~250 g C/m2 lower to 1-m soil depth under exotic than native plantings. Our results indicate that soil carbonate is an active pool and can respond to differences in plant species traits over timescales of years. Significant losses of inorganic C might be avoided by conserving native grasslands in subhumid ecosystems.

Biogeochemical and physical controls on methane fluxes from two ferruginous meromictic lakes.

Meromictic lakes with anoxic bottom waters often have active methane cycles whereby methane is generally produced biogenically under anoxic conditions and oxidized in oxic surface waters prior to reaching the atmosphere. Lakes that contain dissolved ferrous iron in their deep waters (i.e., ferruginous) are rare, but valuable, as geochemical analogues of the conditions that dominated the Earth's oceans during the Precambrian when interactions between the iron and methane cycles could have shaped the greenhouse regulation of the planet's climate. Here, we explored controls on the methane fluxes from Brownie Lake and Canyon Lake, two ferruginous meromictic lakes that contain similar concentrations (max. >1 mM) of dissolved methane in their bottom waters. The order Methanobacteriales was the dominant methanogen detected in both lakes. At Brownie Lake, methanogen abundance, an increase in methane concentration with respect to depths closer to the sediment, and isotopic data suggest methanogenesis is an active process in the anoxic water column. At Canyon Lake, methanogenesis occurred primarily in the sediment. The most abundant aerobic methane-oxidizing bacteria present in both water columns were associated with the Gammaproteobacteria, with little evidence of anaerobic methane oxidizing organisms being present or active. Direct measurements at the surface revealed a methane flux from Brownie Lake that was two orders of magnitude greater than the flux from Canyon Lake. Comparison of measured versus calculated turbulent diffusive fluxes indicates that most of the methane flux at Brownie Lake was non-diffusive. Although the turbulent diffusive methane flux at Canyon Lake was attenuated by methane oxidizing bacteria, dissolved methane was detected in the epilimnion, suggestive of lateral transport of methane from littoral sediments. These results highlight the importance of direct measurements in estimating the total methane flux from water columns, and that non-diffusive transport of methane may be important to consider from other ferruginous systems.