Erik Trampe

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O2 levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.

A Novel Epiphytic Chlorophyll d‐containing Cyanobacterium Isolated from a Mangrove‐associated Red Alga

A new habitat and a new chlorophyll (Chl) d‐containing cyanobacterium belonging to the genus Acaryochloris are reported in this study. Hyperspectral microscopy showed the presence of Chl d‐containing microorganisms in epiphytic biofilms on a red alga (Gelidium caulacantheum) colonizing the pneumato‐phores of a temperate mangrove (Avicennia marina). The presence of Chl d was further proven by high performance liquid chromatography (HPLC)‐based pigment analysis and by confocal imaging of cultured cells. Enrichment of mangrove biofilm samples under near‐infrared radiation (NIR) yielded the new Acaryochloris sp. MPGRS1, which was closely related in terms of 16S rRNA gene sequence to an isolate from the hypertrophic Salton Sea, USA. The new isolate used Chl d as its major photopigment; Chl d and Chl a contents were ~98% and 1%–2% of total cellular chlorophyll, respectively. These findings expand the variety of ecological niches known to harbor Chl d‐containing cyanobacteria and support our working hypothesis that such oxyphototrophs may be ubiquitous in habitats depleted of visible light, but with sufficient NIR exposure.

Microenvironmental Ecology of the Chlorophyll b-Containing Symbiotic Cyanobacterium Prochloron in the Didemnid Ascidian Lissoclinum patella

The discovery of the cyanobacterium Prochloron was the first finding of a bacterial oxyphototroph with chlorophyll (Chl) b, in addition to Chl a. It was first described as Prochloron didemni but a number of clades have since been described. Prochloron is a conspicuously large (7–25 μm) unicellular cyanobacterium living in a symbiotic relationship, primarily with (sub-) tropical didemnid ascidians; it has resisted numerous cultivation attempts and appears truly obligatory symbiotic. Recently, a Prochloron draft genome was published, revealing no lack of metabolic genes that could explain the apparent inability to reproduce and sustain photosynthesis in a free-living stage. Possibly, the unsuccessful cultivation is partly due to a lack of knowledge about the microenvironmental conditions and ecophysiology of Prochloron in its natural habitat. We used microsensors, variable chlorophyll fluorescence imaging and imaging of O2 and pH to obtain a detailed insight to the microenvironmental ecology and photobiology of Prochloron in hospite in the didemnid ascidian Lissoclinum patella. The microenvironment within ascidians is characterized by steep gradients of light and chemical parameters that change rapidly with varying irradiances. The interior zone of the ascidians harboring Prochloron thus became anoxic and acidic within a few minutes of darkness, while the same zone exhibited O2 super-saturation and strongly alkaline pH after a few minutes of illumination. Photosynthesis showed lack of photoinhibition even at high irradiances equivalent to full sunlight, and photosynthesis recovered rapidly after periods of anoxia. We discuss these new insights on the ecological niche of Prochloron and possible interactions with its host and other microbes in light of its recently published genome and a recent study of the overall microbial diversity and metagenome of L. patella.

Fiber-Optic Probes for Small-Scale Measurements of Scalar Irradiance

A new method for producing fiber‐optic microprobes for scalar irradiance (=fluence rate) measurements is described. Such fine‐scale measurements are important in many photobiological disciplines. With the new method, it is possible to cast spherical 30–600 μm wide light integrating sensor tips onto tapered or untapered optical fibers. The sensor tip is constructed by first casting a clear polymethyl methacrylate (PMMA) sphere (~80% of the size of the final probe tip diameter) onto the optical fiber via dip‐coating. Subsequently, the clear sphere is covered with light diffusing layers of PMMA mixed with TiO2 until the fiber probe exhibits a satisfactory isotropic response (typically ±5–10%). We also present an experimental setup for measuring the isotropic response of fiber‐optic scalar irradiance probes in air and water. The fiber probes can be mounted in a syringe equipped with a needle, facilitating retraction of the spherical fiber tip. This makes it, e.g. possible to cut a hole in cohesive tissue with the needle before inserting the probe. The light‐collecting properties of differently sized scalar irradiance probes (30, 40, 100, 300 and 470 μm) produced by this new method were compared to probes produced with previously published methods. The new scalar irradiance probes showed both higher throughput of light, especially for blue light, as well as a better isotropic light collection over a wide spectral range. The new method also allowed manufacturing of significantly smaller scalar irradiance microprobes (down to 30 μm tip diameter) than hitherto possible, and such sensors allow minimally invasive high‐resolution scalar irradiance measurements in thin biofilms, leaves and animal tissues.

Photoregulation in a Kleptochloroplastidic Dinoflagellate, Dinophysis acuta

Some phagotrophic organisms can retain chloroplasts of their photosynthetic prey as so-called kleptochloroplasts and maintain their function for shorter or longer periods of time. Here we show for the first time that the dinoflagellate Dinophysis acuta takes control over “third-hand” chloroplasts obtained from its ciliate prey Mesodinium spp. that originally ingested the cryptophyte chloroplasts. With its kleptochloroplasts, D. acuta can synthesize photosynthetic as well as photoprotective pigments under long-term starvation in the light. Variable chlorophyll fluorescence measurements showed that the kleptochloroplasts were fully functional during 1 month of prey starvation, while the chlorophyll a-specific inorganic carbon uptake decreased within days of prey starvation under an irradiance of 100 μmol photons m-2 s-1. While D. acuta cells can regulate their pigmentation and function of kleptochloroplasts they apparently lose the ability to maintain high inorganic carbon fixation rates.

Kleptoplast photosynthesis is nutritionally relevant in the sea slug Elysia viridis

Several sacoglossan sea slug species feed on macroalgae and incorporate chloroplasts into tubular cells of their digestive diverticula. We investigated the role of the “stolen” chloroplasts (kleptoplasts) in the nutrition of the sea slug Elysia viridis and assessed how their abundance, distribution and photosynthetic activity were affected by light and starvation. Elysia viridis individuals feeding on the macroalga Codium tomentosum were compared with starved specimens kept in dark and low light conditions. A combination of variable Chl a fluorescence and hyperspectral imaging, and HPLC pigment analysis was used to evaluate the spatial and temporal variability of photopigments and of the photosynthetic capacity of kleptoplasts. We show increased loss of weight and body length in dark-starved E. viridis as compared to low light-starved sea slugs. A more pronounced decrease in kleptoplast abundance and lower photosynthetic electron transport rates were observed in dark-starved sea slugs than in low light-starved animals. This study presents strong evidence of the importance of kleptoplast photosynthesis for the nutrition of E. viridis in periods of food scarcity. Deprived of photosynthates, E. viridis could accelerate the breakdown of kleptoplasts in the dark to satisfy its’ energy requirements.

Chlorophyll f distribution and dynamics in cyanobacterial beachrock biofilms.

Chlorophyll (Chl) f, the most far‐red (720–740 nm) absorbing Chl species, was discovered in cyanobacterial isolates from stromatolites and subsequently in other habitats as well. However, the spatial distribution and temporal dynamics of Chl f in a natural habitat have so far not been documented. Here, we report the presence of Chl f in cyanobacterial beachrock biofilms. Hyperspectral imaging on cross‐sections of beachrock from Heron Island (Great Barrier Reef, Australia), showed a strong and widely distributed signature of Chl f absorption in an endolithic layer below the dense cyanobacterial surface biofilm that could be localized to aggregates of Chroococcidiopsis‐like unicellular cyanobacteria packed within a thick common sheath. High‐pressure liquid chromatography‐based pigment analyses showed in situ ratios of Chl f to Chl a of 5% in brown‐pigmented zones of the beachrock, with lower ratios of ~0.5% in the black‐ and pink‐pigmented biofilm zones. Enrichment experiments with black beachrock biofilm showed stimulated synthesis of Chl f and Chl d when grown under near‐infrared radiation (NIR; 740 nm), with a Chl f to Chl a ratio increasing 4‐fold to 2%, whereas the Chl d to Chl a ratio went from 0% to 0.8%. Enrichments grown under white light (400–700 nm) produced no detectable amounts of either Chl d or Chl f. Beachrock cyanobacteria thus exhibited characteristics of far‐red light photoacclimation, enabling Chl f ‐containing cyanobacteria to thrive in optical niches deprived of visible light when sufficient NIR is prevalent.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle

ABSTRACT Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30 μmol H2 liter−1 at about 1-mm depth below the mat surface, slowly decreasing to about 11 μmol H2 liter−1 just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 μmol H2 liter−1, was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H2, it is now possible to study the microdistribution of H2 in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H2 profiles within the mat compared to previous measurements of H2 emission gives much more detailed information about the sources and sinks of H2 in such communities, and it was demonstrated that the high rates of H2 formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H2 is formed as a by-product.

In situ Dynamics of O2, pH, Light, and Photosynthesis in Ikaite Tufa Columns (Ikka Fjord, Greenland)-A Unique Microbial Habitat.

The Ikka Fjord (SW Greenland) harbors a unique microbial habitat in the form of several hundred submarine tufa columns composed of ikaite, a special hexahydrate form of calcium carbonate that precipitates when alkaline phosphate- and carbonate-enriched spring water seeping out of the sea floor meets cold seawater. While several unique heterotrophic microbes have been isolated from the tufa columns, the microbial activity, and the boundary conditions for microbial growth in ikaite have remained unexplored. We present the first detailed in situ characterization of the physico-chemical microenvironment and activity of oxygenic phototrophs thriving within the ikaite columns. In situ underwater microsensor measurements of pH, temperature, and irradiance in the porous ikaite crystal matrix, revealed an extreme microenvironment characterized by low temperatures, strong light attenuation, and gradients of pH changing from pH 9 at the outer column surface to above pH 10 over the first 1–2 cm of the ikaite. This outer layer of the freshly deposited ikaite matrix contained densely pigmented yellow and green zones harboring a diverse phototrophic community dominated by diatoms and cyanobacteria, respectively, as shown by amplicon sequencing. In situ O2 measurements, as well as underwater variable chlorophyll fluorescence measurements of photosynthetic activity, demonstrated high levels of oxygenic photosynthesis in this extreme gradient environment with strong irradiance-driven O2 dynamics ranging from anoxia to hyperoxic conditions in the ikaite matrix, albeit the local formation of gas bubbles buffered the day-night dynamics of O2 in the tufa columns. The microbial phototrophs in the ikaite matrix are embedded in exopolymers forming endolithic biofilms that may interact with mineral formation and cementing of ikaite crystals.