Erik Trampe:
Microenvironmental Ecology of Phototrophs from Extreme Environments

Date: 15-05-2016    Supervisor: Michael Kühl

In the three manuscripts presented in part one of this thesis, I analyse the physicochemical parameters, microenvironmental ecology and species composition of microbial phototrophs in ikaite tufa columns. This work was not easy, and encompassed underwater sampling and microsensor work demanding exceptional skills of the technical diver under challenging conditions during the two fieldtrips to the remote Ikka Fjord in Greenland. These fieldtrips yielded i) the first in-situ mapping of pH and O2 gradients across the outermost two centimetres of the tufa columns, covering the phototrophic growth zone of the ikaite matrix, ii) measurements of diurnal fluctuations in irradiance and O2 under different light scenarios, and iii) the first in situ measurements of photosynthetic activity in the tufa columns (Manuscript 1). This was followed up with more detailed lab-based measurements close to the field site on spatial distribution of major taxonomic groups of phototrophs, detailed measurements of the optical properties of the ikaite matrix, in combination with various microscopic investigations of the phototrophic biofilms in the ikaite (Manuscript 2). Samples brought back for more advanced microscopy in Copenhagen were also used as the initial inocula for enrichments and cultivation of isolates of oxygenic phototrophs from the ikaite columns. Most of the culture work was conducted in the lab of Prof. Richard Castenholz at University of Oregon, OR, USA. Many isolates of phototrophs were obtained, several of which have now been tentatively identified taxonomically, and their phylogenetic relationships been evaluated (Manuscript 3).

Part 2 of the thesis includes three manuscripts that focus on cyanobacteria that have evolved special photopigments in contrast to other cyanobacteria. In Manuscript 4 we show the occurrence of a novel epiphytic Acaryochloris sp. in biofilms associated with a red algae growing on temperate mangrove roots, giving further evidence for more global distribution of Chl d-containing cyanobacteria than previously thought. In Manuscript 5, the microenvironmental ecology and diversity of three distinct biofilm communities associated with the didemnid ascidian Lissoclinum platella was evaluated by means of microsensor measurements of O2 and scalar irradiance, hyperspectral imaging of pigment distribution, variable chlorophyll fluorescence assessment of relative PSII electron transport rates, and amplicon sequencing of the ascidian microbiome. We found a depth related distribution pattern of Acaryochloris and other far-red light adapted phototrophs, driven by the increased attenuation of far red light in deeper waters. In Manuscript 6, the ecophysiology of the cyanobacteria under in hospite conditions in L. platella was assessed revealing dynamic irradiancedriven changes in O2, pH and photosynthesis using a suite of advanced bioimaging methods for variable chlorophyll fluorescence imaging, and planar optode imaging of pH and O2 distribution in the ascidian and its associated internal and external biofilms.

Part 3 of the thesis consists of two manuscripts, Manuscript 7, and 8. In manuscript 7 we investigated the microenvironmental ecology of the beachrock on Heron Island, GBR, Australia. We obtained in-situ environmental parameters of, temperature, irradiance and salinity on the beachrock platform, and analyzed physico-chemical properties of the microenvironment by means of microsensor measurements of fine scale scalar irradiance, O2 and pH, in combination with HPLC pigment analysis and assessment of photosynthetic performance of the cyanobacterial biofilms colonizing the Black and Pink zones. Further laboratory measurements included imaging of planar O2 optodes for oxygen evolution. A catalog of several cyanobacterial community members was tentatively assigned to the two distinctly colored beachrock zones by Scanning Electron Microscopy. In Manuscript 8, we present the discovery of Chl f in cyanobacterial biofilms growing on beachrock, Chl f was found in unicellular cyanobacteria thriving in a light limited habitat below a densely pigmented surface biofilm of other cyanobacteria, and the relative amount of Chl f to Chl a could be further enhanced by incubating beachrock samples under NIR indicating the potential for dynamic photoacclimation to far-red light in the cyanobacterial beachrock biofilms.

Part 4 of the thesis contains one manuscript, Manuscript 9. In this study, we investigated the potential for photoregulation in the kleptoplastidic dinoflagellate Dinophysis acuta utilizing a broad array of methods. Kleptoplasdity is a process where a host consumes the chloroplast-containing organism and digest most but the chloroplasts, and continues to gain photosynthates from these “stolen” chloroplasts for a period of time. We analyzed D. acuta cells during a starvation experiment by means of growth rates, microscopic imaging of photosynthetic capacity, localization of kleptochloroplasts, inorganic carbon uptake, and tracking of fluctuations in pigment content by spectrophotometric and HPLC analysis. We found that D. acuta cells can regulate the pigmentation and function of their kleptochloroplasts but that they apparently lose the ability to maintain high inorganic carbon fixation rates.

The last part of the thesis contains three manuscripts, Manuscript 10, 11 and 12, that represent methodical studies. Some of the work included in Manuscript 10, was performed but not completely analyzed and finalized in part of my master thesis and presents a new multicolor (red, green, and blue excitation light) system for microscopic imaging of variable chlorophyll fluorescence. In addition to a thorough description of the commercially available imaging system, we developed an additional custom build temperature controlled microscope slide mount, and we applied the system in a number of experiments demonstrating the functionalities and capabilities of the instrumentation.

In Manuscript 11, we compared three different, but widely used methods of estimating photosynthetic activity, namely measurements of O2 evolution, 14C assimilation, and variable chlorophyll fluorescence imaging. All techniques were employed in a light acclimation experiment with the diatom Coscinodiscus granii. Each of the applied methods evaluates different parts and reactions in the photosynthetic apparatus, which makes a direct quantitative comparison of rates difficult, although different degrees of correlation were found between all three methods. When used in combination, they provide information about the internal relationship of photosynthetic pathways as well as the variation in photosynthetic capacity between individual cells within a single algal culture.

In Manuscript 12, we describe a new method for producing fiber-optic scalar irradiance microprobes with tip diameters of 30-600 μm. 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 in comparison to other scalar irradiance probes. The new method also allowed manufacturing of significantly smaller scalar irradiance microprobes than hitherto possible, which facilitates less invasive scalar irradiance measurements in a wide array of organisms and materials.