Annual Cycle of Mat-Forming Filamentous Alga Tribonema cf. minus (Stramenopiles, Xanthophyceae) in Hydro-Terrestrial Habitats in the High Arctic Revealed By Multiparameter Fluorescent Staining.

The filamentous microalga Tribonema sp. (Stramenopiles, Xanthophyceae) plays an important role in shallow water polar (streams and seepages) and seasonally cold habitats in temperate regions (ponds). In these habitats, freezing and desiccation, and thus freeze-thawing and drying-rewetting cycles, are frequent. These regions produce visible biomass and are important components of low temperature-adapted communities. We characterized the annual cycles of a Tribonema cf. minus population in two habitats (seepage and stream) in the High Arctic, Svalbard. Seasonality, locality, and their combination (particularly changing environmental conditions) together with cultivation conditions of strains significantly affected their morphological characteristics. Morphological changes following hardening processes related to preparation for the winter period (transition from vegetative cells to akinete and/or pre-akinete) were recorded. Over the year, positive water temperatures (warmest 13.3°C) occurred for 5 months while negative (lowest temperature was -17.4°C) lasted for 7 months. In winter, there were two melt periods. Vitality staining protocol showed a high number of viable (77.4% and 53.8%) and dormant cells (1.7% and 4.1%; capable of growth and reproduction once suitable conditions return) in the winter seepage and stream, respectively. NPQ and OJIP chlorophyll fluorescence parameters revealed several hours recovery of photosynthesis (both field and control samples). During recovery, only minor or mild stress on photosynthesis was detected. FV /FM values (the photosynthetic efficiency of photosystem II in a dark-adapted state) in all field and control samples varied around 0.4. Tribonema cf. minus is capable of surviving winter Arctic conditions (perennial strategy).

Response of short-term heat shock on photosynthetic activity of soil crust cyanobacteria.

Short-term heat exposure in tropical regions can generate severe stress in the photosynthetic activity of soil crust cyanobacteria. We investigated the responses of two filamentous cyanobacteria, Scytonema tolypothrichoides and Tolypothrix bouteillei, to 1hr exposure at 35, 45, and 55 °C using variable chlorophyll fluorescence. Protocols for maximum quantum yield (FV/FM) and dark recovery of chlorophyll a fluorescence (OJIP) transient were applied. Heat exposure caused damage to the donor side of PSII, indicated by a decrease in FV/FM and a rapid increase in F0. After heat stress, photochemical energy utilization (φPo, φETo, and φRE1o) declined and energy dissipation (φDIo) increased. At 45 °C, the photosynthetic apparatus was reversibly damaged, since full recovery was observed after 7 days of relaxation. S. tolypothrichoides was more resistant to heat stress than T. bouteillei, confirming better adaptation to higher temperatures as observed in growth experiments.

Ecophysiological Features of Polar Soil Unicellular Microalgae1.

Due to their ecological, physiological, and molecular adaptations to low and varying temperatures, as well as varying seasonal irradiances, polar non-marine eukaryotic microalgae could be suitable for low-temperature biotechnology. Adaptations include the synthesis of compounds from different metabolic pathways that protect them against stress. Production of biological compounds and various biotechnological applications, for instance, water treatment technology, are of interest to humans. To select prospective strains for future low-temperature biotechnology in polar regions, temperature and irradiance of growth requirements (Q10 and Ea of 10 polar soil unicellular strains) were evaluated. In terms of temperature, three groups of strains were recognized: (i) cold-preferring where temperature optima ranged between 10.1 and 18.4°C, growth rate 0.252 and 0.344 · d-1 , (ii) cold- and warm-tolerating with optima above 10°C and growth rate 0.162-0.341 · d-1 , and (iii) warm-preferring temperatures above 20°C and growth rate 0.249-0.357 · d-1 . Their light requirements were low. Mean values Q10 for specific growth rate ranged from 0.7 to 3.1. The lowest Ea values were observed on cold-preferring and the highest in the warm-preferring strains. One strain from each temperature group was selected for PN and RD measurements. The PN :RD ratio of the warm-preferring strains was less affected by temperature similarly as Q10 and Ea. For future biotechnological applications, the strains with broad temperature tolerance (i.e., the group of cold- and warm-tolerating and warm-preferring strains) will be most useful.

Estimation of growth and exopolysaccharide production by two soil cyanobacteria, Scytonema tolypothrichoides and Tolypothrix bouteillei as determined by cultivation in irradiance and temperature crossed gradients.

Two filamentous cyanobacteria of the genera Scytonema and Tolypothrix were reported to be effective for stabilizing soil in arid areas due to the production of significant amounts of extracellular polysaccharides (EPS). These EPS may also have applications in the biotechnology industry. Therefore, two cyanobacterial species, Scytonema tolypothrichoides and Tolypothrix bouteillei were examined using crossed gradients of temperature (8-40°C) and irradiance (3-21 W m-2) to identify their temperature and irradiance optima for maximum biomass and EPS production. According to their reported temperature requirements, both strains were considered mesophilic. The optimum growth range of temperature in S. tolypothrichoides (27 to 34°C) was higher than T. bouteillei (22-32°C). The optimum irradiance range for growth of S. tolypothrichoides (9-13 W m-2) was slightly lower than T. bouteillei (7-18 W m-2). Maximum EPS production by S. tolypothrichoides occurred at similar temperatures (28-34°C) as T. bouteillei (27-34°C), both slightly higher than for maximum growth. The optimum irradiance range for EPS production was comparable to that for growth in S. tolypotrichoides (8-13 W m-2), and slightly lower in T. bouteillei (7-17 W m-2). The Redundancy Analysis confirmed that temperature was the most important controlling factor and protocols for field applications or for mass cultivation can now be developed.

The green alga Dictyosphaerium chlorelloides biomass and polysaccharides production determined using cultivation in crossed gradients of temperature and light.

The green microalga Dictyosphaerium chlorelloides was identified as promising microorganism for biotechnological production of exopolysaccharides (EPS). In stationary phase the culture suspension solidifies to thick gel, with very high viscosity and high content of EPS which may be interesting for many biotechnological applications. To develop cultivation protocol for maximum biomass/polysaccharide production, the optimum conditions for growth and polysaccharides production were determined in this study using the crossed gradient cultivation method. Temperature and irradiance requirements of Dictyosphaerium chlorelloides were evaluated by statistical analyses for growth rate/biomass, extracellular (EPS) and intracellular (IPS) polysaccharides contents in crossed gradients of temperature (4-45°C) and irradiance (2-18 W/m2, 9.1 - 82.3 µmol/(m2 s)). The maximum relative growth rate was observed at temperatures around 19.2°C and relatively low irradiances in range 2.6-11 W/m2 (11.9-50.3 µmol/(m2 s)). The maximum IPS production was observed at temperatures around 19.2°C and irradiance around 11 W/m2 (50.3 µmol/(m2 s)). The maximum production of EPS was observed at temperatures around 25.7°C and similar irradiances as IPS production. Due to temperature separation of growth and EPS production, development of cultivation protocol based controlled temperature manipulation is possible.

Identity, ecology and ecophysiology of planktic green algae dominating in ice-covered lakes on James Ross Island (northeastern Antarctic Peninsula).

The aim of this study was to assess the phylogenetic relationships, ecology and ecophysiological characteristics of the dominant planktic algae in ice-covered lakes on James Ross Island (northeastern Antarctic Peninsula). Phylogenetic analyses of 18S rDNA together with analysis of ITS2 rDNA secondary structure and cell morphology revealed that the two strains belong to one species of the genus Monoraphidium (Chlorophyta, Sphaeropleales, Selenastraceae) that should be described as new in future. Immotile green algae are thus apparently capable to become the dominant primary producer in the extreme environment of Antarctic lakes with extensive ice-cover. The strains grew in a wide temperature range, but the growth was inhibited at temperatures above 20 °C, indicating their adaptation to low temperature. Preferences for low irradiances reflected the light conditions in their original habitat. Together with relatively high growth rates (0.4-0.5 day-1) and unprecedently high content of polyunsaturated fatty acids (PUFA, more than 70% of total fatty acids), it makes these isolates interesting candidates for biotechnological applications.

Platinum anniversary: virus and lichen alga together more than 70 years.

Trebouxia aggregata (Archibald) Gärtner (phylum Chlorophyta, family Trebouxiaceae), a lichen symbiotic alga, has been identified as host of the well-known herbaceous plant virus Cauliflower mosaic virus (CaMV, family Caulimoviridae). The alga had been isolated from Xanthoria parietina more than 70 years ago and has been maintained in a collection since that time. The CaMV detected in this collection entry has now been completely sequenced. The virus from T. aggregata is mechanically transmissible to a herbaceous host and induces disease symptoms there. Its genome differs by 173 nt from the closest European CaMV-D/H isolate from cauliflower. No site under positive selection was found on the CaMV genome from T. aggregata. We therefore assume that the virus's presence in this alga was not sufficiently long to fix any specific changes in its genome. Apart from this symbiotic alga, CaMV capsid protein sequences were amplified from many other non-symbiotic algae species maintained in a collection (e.g., Oonephris obesa, Elliptochloris sp., Microthamnion kuetzingianum, Chlorella vulgaris, Pseudococcomyxa sp.). CaMV-free Chlorella vulgaris was treated with CaMV to establish virus infection. The virus was still detected there after five passages. The virus infection is morphologically symptomless on Chlorella algae and the photosynthesis activity is slightly decreased in comparison to CaMV-free alga culture. This is the first proof as to the natural presence of CaMV in algae and the first demonstration of algae being artificially infected with this virus.

The ice nucleation activity of extremophilic algae.

Differences in the level of cold acclimation and cryoprotection estimated as ice nucleation activity in snow algae (Chlamydomonas cf. nivalis and Chloromonas nivalis), lichen symbiotic algae (Trebouxia asymmetrica, Trebouxia erici and Trebouxia glomerata), and a mesophilic strain (Chlamydomonas reinhardti) were evaluated. Ice nucleation activity was measured using the freezing droplet method. Measurements were performed using suspensions of cells of A750 (absorbance at 750 nm) ~ 1, 0.1, 0.01 and 0.001 dilutions for each strain. The algae had lower ice nucleation activity, with the exception of Chloromonas nivalis contaminated by bacteria. The supercooling points of the snow algae were higher than those of lichen photobionts. The supercooling points of both, mesophilic and snow Chlamydomonas strains were similar. The lower freezing temperatures of the lichen algae may reflect either the more extreme and more variable environmental conditions of the original localities or the different cellular structure of the strains examined.

A Laboratory of Extremophiles: Iceland Coordination Action for Research Activities on Life in Extreme Environments (CAREX) Field Campaign.

Existence of life in extreme environments has been known for a long time, and their habitants have been investigated by different scientific disciplines for decades. However, reports of multidisciplinary research are uncommon. In this paper, we report an interdisciplinary three-day field campaign conducted in the framework of the Coordination Action for Research Activities on Life in Extreme Environments (CAREX) FP7EU program, with participation of experts in the fields of life and earth sciences. In situ experiments and sampling were performed in a 20 m long hot springs system of different temperature (57 °C to 100 °C) and pH (2 to 4). Abiotic factors were measured to study their influence on the diversity. The CO2 and H2S concentration varied at different sampling locations in the system, but the SO2 remained the same. Four biofilms, mainly composed by four different algae and phototrophic protists, showed differences in photosynthetic activity. Varying temperature of the sampling location affects chlorophyll fluorescence, not only in the microbial mats, but plants (Juncus), indicating selective adaptation to the environmental conditions. Quantitative polymerase chain reaction (PCR), DNA microarray and denaturing gradient gel electrophoresis (DGGE)-based analysis in laboratory showed the presence of a diverse microbial population. Even a short duration (30 h) deployment of a micro colonizer in this hot spring system led to colonization of microorganisms based on ribosomal intergenic spacer (RISA) analysis. Polyphasic analysis of this hot spring system was possible due to the involvement of multidisciplinary approaches.

Photochemical performance of the acidophilic red alga Cyanidium sp. in a pH gradient.

The acidophilic red alga Cyanidium sp. is one of the dominant mat-forming species in the highly acidic waters of Río Tinto, Spain. The culture of Cyanidium sp., isolated from a microbial mat sample collected at Río Tinto, was exposed to 9 different pH conditions in a gradient from 0.5 to 5 for 24 h and its physiological status evaluated by variable chlorophyll a fluorescence kinetics measurements. Maximum quantum yield was determined after 30 min, 1 h, 2 h, 4 h, 6 h and 24 h of exposure after 15 min dark adaptation. The effect of pH on photochemical activity of Cyanidium sp. was observable as early as 30 min after exposure and the pattern remained stable or with only minor modifications for 24 h. The optimum pH ranged from 1.5 to 2.5. A steep decrease of the photochemical activity was observed at pH below 1 even after 30 min of exposure. Although the alga had tolerated the exposure to pH = 1 for at least 6 h, longer (24 h) exposure resulted in reduction of the photochemical activity. At pH above 2.5, the decline was more moderate and its negative effect on photochemistry was less severe. According to the fluorescence measurements, the red alga Cyanidium sp. is well-adapted to prevailing pH at its original locality at Río Tinto, i.e. pH of 1 to 3. The short-term survival in pH < 1.5 may be adaptation to rare exposures to such low pH in the field. The tolerance of pH above 3 could be caused by adaptation to the microenvironment of the inner parts of microbial mats in which Cyanidium sp. usually dominates and where higher pH could occur due to photosynthetic oxygen production.

Rapid algal toxicity assay using variable chlorophyll fluorescence for Chlorella kessleri (chlorophyta).

Three methods of algal assays--the standard assay, microassay, and the proposed fluorescence assay--are compared from the point of view of reliability of EC50 detection, the minimum required time for the detection, sensitivity of individual measurement, i.e. at which cell density the particular assay can be used for EC50 estimation, and the time stability of the EC50 values. The assays were performed with green alga Chlorella kessleri strain LARG/1 growing in potassium dichromate solution in Z-medium ranging from 0.01 to 100 mg Cr L⁻¹. The inoculation cell density was set according to the standards to 104 cells mL⁻¹ and according to spectrophotometer/plate reader detection limit. The average EC50 ranged from 0.096 to 0.649 mg Cr L⁻¹ and there were no significant differences in EC50 between the assay type and the inoculation methods with the exception of the significant difference between EC(c)5072 (EC50 established from biomass measured as chlorophyll a concentration after 72 h of cultivation) in the standard assay and EC(r)50 (EC50 derived from growth rate) in the microassay in the standard inoculation experiment due to low variability of their values. The EC(f)50 (EC50 derived from variable fluorescence measurement) values correspond to EC50 values derived from the growth rates. Fluorescence measurement revealed the toxic effect of the chromium after 24 h of exposure at cell density of 5 x 104 cells mL⁻¹, less by half than other used assay methods. The positive correlation of EC(f)50 and time was found in the standard inoculation experiment but opposite effect was observed at the spectrophotometric one.