Sten Littmann

Methane oxidation coupled to oxygenic photosynthesis in anoxic waters

Freshwater lakes represent large methane sources that, in contrast to the Ocean, significantly contribute to non-anthropogenic methane emissions to the atmosphere. Particularly mixed lakes are major methane emitters, while permanently and seasonally stratified lakes with anoxic bottom waters are often characterized by strongly reduced methane emissions. The causes for this reduced methane flux from anoxic lake waters are not fully understood. Here we identified the microorganisms and processes responsible for the near complete consumption of methane in the anoxic waters of a permanently stratified lake, Lago di Cadagno. Interestingly, known anaerobic methanotrophs could not be detected in these waters. Instead, we found abundant gamma-proteobacterial aerobic methane-oxidizing bacteria active in the anoxic waters. In vitro incubations revealed that, among all the tested potential electron acceptors, only the addition of oxygen enhanced the rates of methane oxidation. An equally pronounced stimulation was also observed when the anoxic water samples were incubated in the light. Our combined results from molecular, biogeochemical and single-cell analyses indicate that methane removal at the anoxic chemocline of Lago di Cadagno is due to true aerobic oxidation of methane fuelled by in situ oxygen production by photosynthetic algae. A similar mechanism could be active in seasonally stratified lakes and marine basins such as the Black Sea, where light penetrates to the anoxic chemocline. Given the widespread occurrence of seasonally stratified anoxic lakes, aerobic methane oxidation coupled to oxygenic photosynthesis might have an important but so far neglected role in methane emissions from lakes.

Light-Dependent Aerobic Methane Oxidation Reduces Methane Emissions from Seasonally Stratified Lakes.

Lakes are a natural source of methane to the atmosphere and contribute significantly to total emissions compared to the oceans. Controls on methane emissions from lake surfaces, particularly biotic processes within anoxic hypolimnia, are only partially understood. Here we investigated biological methane oxidation in the water column of the seasonally stratified Lake Rotsee. A zone of methane oxidation extending from the oxic/anoxic interface into anoxic waters was identified by chemical profiling of oxygen, methane and δ13C of methane. Incubation experiments with 13C-methane yielded highest oxidation rates within the oxycline, and comparable rates were measured in anoxic waters. Despite predominantly anoxic conditions within the zone of methane oxidation, known groups of anaerobic methanotrophic archaea were conspicuously absent. Instead, aerobic gammaproteobacterial methanotrophs were identified as the active methane oxidizers. In addition, continuous oxidation and maximum rates always occurred under light conditions. These findings, along with the detection of chlorophyll a, suggest that aerobic methane oxidation is tightly coupled to light-dependent photosynthetic oxygen production both at the oxycline and in the anoxic bottom layer. It is likely that this interaction between oxygenic phototrophs and aerobic methanotrophs represents a widespread mechanism by which methane is oxidized in lake water, thus diminishing its release into the atmosphere.

The small unicellular diazotrophic symbiont, UCYN-A, is a key player in the marine nitrogen cycle.

Microbial dinitrogen (N2) fixation, the nitrogenase enzyme-catalysed reduction of N2 gas into biologically available ammonia, is the main source of new nitrogen (N) in the ocean. For more than 50 years, oceanic N2 fixation has mainly been attributed to the activity of the colonial cyanobacterium Trichodesmium1,2. Other smaller N2-fixing microorganisms (diazotrophs)—in particular the unicellular cyanobacteria group A (UCYN-A)—are, however, abundant enough to potentially contribute significantly to N2 fixation in the surface waters of the oceans3–6. Despite their abundance, the contribution of UCYN-A to oceanic N2 fixation has so far not been directly quantified. Here, we show that in one of the main areas of oceanic N2 fixation, the tropical North Atlantic7, the symbiotic cyanobacterium UCYN-A contributed to N2 fixation similarly to Trichodesmium. Two types of UCYN-A, UCYN-A1 and -A2, were observed to live in symbioses with specific eukaryotic algae. Single-cell analyses showed that both algae–UCYN-A symbioses actively fixed N2, contributing ∼20% to N2 fixation in the tropical North Atlantic, revealing their significance in this region. These symbioses had growth rates five to ten times higher than Trichodesmium, implying a rapid transfer of UCYN-A-fixed N into the food web that might significantly raise their actual contribution to N2 fixation. Our analysis of global 16S rRNA gene databases showed that UCYN-A occurs in surface waters from the Arctic to the Antarctic Circle and thus probably contributes to N2 fixation in a much larger oceanic area than previously thought. Based on their high rates of N2 fixation and cosmopolitan distribution, we hypothesize that UCYN-A plays a major, but currently overlooked role in the oceanic N cycle.

N2-fixation, ammonium release and N-transfer to the microbial and classical food web within a plankton community

We investigated the role of N2-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with 15N2, Aphanizomenon spp. showed a strong 15N-enrichment implying substantial 15N2-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of 15NH4+ from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH4+ fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N2-fixation within colonies. No N2-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH4+ uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N2 as NH4+. However, NH4+ did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N2 as NH4+, which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N2-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected 13C- and 15N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate 13C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.

Phenotypic heterogeneity driven by nutrient limitation promotes growth in fluctuating environments

Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity1 in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future2–9. It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4+ limitation shapes phenotypic heterogeneity in N2 fixation. In turn, the N2 fixation rate of single cells during NH4+ limitation correlates positively with their growth rate after a shift to NH4+ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges—nutrient limitation and fluctuations—that many microorganisms face.

Crenothrix are major methane consumers in stratified lakes

Methane-oxidizing bacteria represent a major biological sink for methane and are thus Earth’s natural protection against this potent greenhouse gas. Here we show that in two stratified freshwater lakes a substantial part of upward-diffusing methane was oxidized by filamentous gamma-proteobacteria related to Crenothrix polyspora. These filamentous bacteria have been known as contaminants of drinking water supplies since 1870, but their role in the environmental methane removal has remained unclear. While oxidizing methane, these organisms were assigned an ‘unusual’ methane monooxygenase (MMO), which was only distantly related to ‘classical’ MMO of gamma-proteobacterial methanotrophs. We now correct this assignment and show that Crenothrix encode a typical gamma-proteobacterial PmoA. Stable isotope labeling in combination swith single-cell imaging mass spectrometry revealed methane-dependent growth of the lacustrine Crenothrix with oxygen as well as under oxygen-deficient conditions. Crenothrix genomes encoded pathways for the respiration of oxygen as well as for the reduction of nitrate to N2O. The observed abundance and planktonic growth of Crenothrix suggest that these methanotrophs can act as a relevant biological sink for methane in stratified lakes and should be considered in the context of environmental removal of methane.

Environmental Breviatea harbour mutualistic Arcobacter epibionts

Summary Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi1–3. This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle4. Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments showed that the association of L. limosa with Arcobacter was driven by the transfer of hydrogen and was mutualistic, providing benefits to both partners. With whole genome sequencing and differential proteomics we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H accepting hydrogenase, which was expressed in the presence, but not in the absence of Arcobacter. Differential proteomics further revealed that the presence of Lenisia stimulated expression of known “virulence” factors by Arcobacter. These proteins typically enable colonization of animal cells during infection5, but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic datasets of other Breviatea4 revealed the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism6 as shown here for Arcobacter and Breviatea.

Use of carbon monoxide and hydrogen by a bacteria–animal symbiosis from seagrass sediments

Summary The gutless marine worm O lavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO 2) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O . algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H 2) might also be used as energy sources. We provide direct evidence that the O . algarvensis symbiosis consumes CO and H 2. Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3‐symbiont, uses the energy from CO oxidation to fix CO 2. Pore water analysis revealed considerable in‐situ concentrations of CO and H 2 in the O . algarvensis environment, Mediterranean seagrass sediments. Pore water H 2 concentrations (89–2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36‐fold higher than previously known from shallow‐water marine sediments. Pore water CO concentrations (17–51 nM) were twice as high as in the overlying seawater (no literature data from other shallow‐water sediments are available for comparison). Ex‐situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.

Identification and activity of acetate-assimilating bacteria in diffuse fluids venting from two deep-sea hydrothermal systems

Diffuse hydrothermal fluids often contain organic compounds such as hydrocarbons, lipids, and organic acids. Microorganisms consuming these compounds at hydrothermal sites are so far only known from cultivation-dependent studies. To identify potential heterotrophs without prior cultivation, we combined microbial community analysis with short-term incubations using (13)C-labeled acetate at two distinct hydrothermal systems. We followed cell growth and assimilation of (13)C into single cells by nanoSIMS combined with fluorescence in situ hybridization (FISH). In 55 °C-fluids from the Menez Gwen hydrothermal system/Mid-Atlantic Ridge, a novel epsilonproteobacterial group accounted for nearly all assimilation of acetate, representing the first aerobic acetate-consuming member of the Nautiliales. In contrast, Gammaproteobacteria dominated the (13) C-acetate assimilation in incubations of 37 °C-fluids from the back-arc hydrothermal system in the Manus Basin/Papua New Guinea. Here, 16S rRNA gene sequences were mostly related to mesophilic Marinobacter, reflecting the high content of seawater in these fluids. The rapid growth of microorganisms upon acetate addition suggests that acetate consumers in diffuse fluids are copiotrophic opportunists, which quickly exploit their energy sources, whenever available under the spatially and temporally highly fluctuating conditions. Our data provide first insights into the heterotrophic microbial community, catalyzing an under-investigated part of microbial carbon cycling at hydrothermal vents.