Strong and widespread cycloheximide resistance in Stichococcus-like eukaryotic algal taxa.

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) ("Antarctica" 1 and "Antarctica" 2), one isolated from this island about 50 years ago ("Antarctica" 3) and single Arctic ("Arctic"), temperate ("Temperate") and tropical ("Tropical") strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except "Temperate" were highly resistant to CHX (MIC > 1000 µg/mL), while "Temperate" was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, "Temperate", was confirmed to be S. bacillaris, while "Tropical" represents the newly erected genus Tetratostichococcus, "Antarctica 1" Tritostichococcus, and "Antarctica 2", "Antarctica 3" and "Arctic" Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.

Enhancing microalgal photosynthesis and productivity in wastewater treatment high rate algal ponds for biofuel production.

With microalgal biofuels currently receiving much attention, there has been renewed interest in the combined use of high rate algal ponds (HRAP) for wastewater treatment and biofuel production. This combined use of HRAPs is considered to be an economically feasible option for biofuel production, however, increased microalgal productivity and nutrient removal together with reduced capital costs are needed before it can be commercially viable. Despite HRAPs being an established technology, microalgal photosynthesis and productivity is still limited in these ponds and is well below the theoretical maximum. This paper critically evaluates the parameters that limit microalgal light absorption and photosynthesis in wastewater HRAPs and examines biological, chemical and physical options for improving light absorption and utilisation, with the view of enhancing biomass production and nutrient removal.

The effects of CO2 addition along a pH gradient on wastewater microalgal photo-physiology, biomass production and nutrient removal.

Carbon limitation in domestic wastewater high rate algal ponds is thought to constrain microalgal photo-physiology and productivity, particularly in summer. This paper investigates the effects of CO2 addition along a pH gradient on the performance of wastewater microalgae in high rate algal mesocosms. Performance was measured in terms of light absorption, electron transport rate, photosynthetic efficiency, biomass production and nutrient removal efficiency. Light absorption by the microalgae increased by up to 128% with increasing CO2 supply, while a reduction in the package effect meant that there was less internal self-shading thereby increasing the efficiency of light absorption. CO2 augmentation increased the maximum rate of both electron transport and photosynthesis by up to 256%. This led to increased biomass, with the highest yield occurring at the highest dissolved inorganic carbon/lowest pH combination tested (pH 6.5), with a doubling of chlorophyll-a (Chl-a) biomass while total microalgal biovolume increased by 660% in Micractinium bornhemiense and by 260% in Pediastrum boryanum dominated cultures. Increased microalgal biomass did not off-set the reduction in ammonia volatilisation in the control and overall nutrient removal was lower with CO2 than without. Microalgal nutrient removal efficiency decreased as pH decreased and may have been related to decreased Chl-a per cell. This experiment demonstrated that CO2 augmentation increased microalgal biomass in two distinct communities, however, care must be taken when interpreting results from standard biomass measurements with respect to CO2 augmentation.

Modifying the high rate algal pond light environment and its effects on light absorption and photosynthesis.

The combined use of high rate algal ponds (HRAPs) for wastewater treatment and commercial algal production is considered to be an economically viable option. However, microalgal photosynthesis and biomass productivity is constrained in HRAPs due to light limitation. This paper investigates how the light climate in the HRAP can be modified through changes in pond depth, hydraulic retention time (HRT) and light/dark turnover rate and how this impacts light absorption and utilisation by the microalgae. Wastewater treatment HRAPs were operated at three different pond depth and HRT during autumn. Light absorption by the microalgae was most affected by HRT, significantly decreasing with increasing HRT, due to increased internal self-shading. Photosynthetic performance (as defined by Pmax, Ek and α), significantly increased with increasing pond depth and decreasing HRT. Despite this, increasing pond depth and/or HRT, resulted in decreased pond light climate and overall integrated water column net oxygen production. However, increased light/dark turnover was able to compensate for this decrease, bringing the net oxygen production in line with shallower ponds operated at shorter HRT. On overcast days, modelled daily net photosynthesis significantly increased with increased light/dark turnover, however, on clear days such increased turnover did not enhance photosynthesis. This study has showed that light absorption and photosynthetic performance of wastewater microalgae can be modified through changes to pond depth, HRT and light/dark turnover.

Effects of two different nutrient loads on microalgal production, nutrient removal and photosynthetic efficiency in pilot-scale wastewater high rate algal ponds.

When wastewater treatment high rate algal ponds (HRAP) are coupled with resource recovery processes, such as biofuel production, short hydraulic retention times (HRTs) are often favoured to increase the microalgal biomass productivity. However, short HRT can result in increased nutrient load to the HRAP which may negatively impact on the performance of the microalgae. This paper investigate the effects of high (NH4-N mean concentration 39.7 ± 17.9 g m(-3)) and moderate ((NH4-N mean concentration 19.9 ± 8.9 g m(-3)) nutrient loads and short HRT on the performance of microalgae with respect to light absorption, photosynthesis, biomass production and nutrient removal in pilot-scale (total volume 8 m(3)) wastewater treatment HRAPs. Microalgal biomass productivity was significantly higher under high nutrient loads, with a 133% and 126% increase in the chlorophyll-a and VSS areal productivities, respectively. Microalgae were more efficient at assimilating NH4-N from the wastewater under higher nutrient loads compared to moderate loads. Higher microalgal biomass with increased nutrient load resulted in increased light attenuation in the HRAP and lower light absorption efficiency by the microalgae. High nutrient loads also resulted in improved photosynthetic performance with significantly higher maximum rates of electron transport, oxygen production and quantum yield. This experiment demonstrated that microalgal productivity and nutrient removal efficiency were not inhibited by high nutrient loads, however, higher loads resulted in lower water quality in effluent discharge.

Wastewater microalgal production, nutrient removal and physiological adaptation in response to changes in mixing frequency.

Laminar flows are a common problem in high rate algal ponds (HRAP) due to their long channels and gentle mixing by a single paddlewheel. Sustained laminar flows may modify the amount of light microalgal cells are exposed to, increase the boundary layer between the cell and the environment and increase settling out of cells onto the pond bottom. To date, there has been little focus on the effects of the time between mixing events (frequency of mixing) on the performance of microalgae in wastewater treatment HRAPs. This paper investigates the performance of three morphologically distinct microalgae in wastewater treatment high rate algal mesocosms operated at four different mixing frequencies (continuous, mixed every 45 min, mixed every 90 min and no mixing). Microalgal performance was measured in terms of biomass concentration, nutrient removal efficiency, light utilisation and photosynthetic performance. Microalgal biomass increased significantly with increasing mixing frequency for the two colonial species but did not differ for the single celled species. All three species were more efficient at NH4-N uptake as the frequency of mixing increased. Increased frequency of mixing supported larger colonies with improved harvest-ability by gravity but at the expense of efficient light absorption and maximum rate of photosynthesis. However, maximum quantum yield was highest in the continuously mixed cultures due to higher efficiency of photosynthesis under light limited conditions. Based on these results, higher microalgal productivity, improved wastewater treatment and better gravity based harvest-ability can be achieved with the inclusion of more mixing points and reduced laminar flows in full-scale HRAP.

Diverse small circular single-stranded DNA viruses identified in a freshwater pond on the McMurdo Ice Shelf (Antarctica).

Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904-3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica.

An estuarine species of the alga Vaucheria (Xanthophyceae) displays an increased capacity for turgor regulation when compared to a freshwater species.

Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Apart from a few studies on freshwater oomycetes, the ability of stramenopiles to turgor regulate has not been investigated. In this study, turgor regulation and growth were compared in two species of the stramenopile alga Vaucheria, Vaucheria erythrospora isolated from an estuarine habitat, and Vaucheria repens isolated from a freshwater habitat. Species were identified using their rbcL sequences and respective morphologies. Using a single cell pressure probe to directly measure turgor in Vaucheria after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were >0.5 MPa for V. erythrospora and <0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Turgor recovery in V. erythrospora was inhibited by Gd(3+) and TEA, suggesting a role for mechanosensitive channels, nonselective cation channels, and K(+) channels in the process. Growth studies showed that V. erythrospora was able to grow over a wider range of NaCl concentrations. These responses may underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater. The fact that both species can turgor regulate may indicate a fundamental difference between members of the Stramenopila, as research to date on oomycetes suggests they are unable to turgor regulate.

MOLECULAR PHYLOGENY OF ANTARCTIC PRASIOLA (PRASIOLALES, TREBOUXIOPHYCEAE) REVEALS EXTENSIVE CRYPTIC DIVERSITY(1).

Trebouxiophytes of the genus Prasiola are well known in Antarctica, where they are among the most important primary producers. Although many aspects of their biology have been thoroughly investigated, the scarcity of molecular data has so far prevented an accurate assessment of their taxonomy and phylogenetic position. Using sequences of the chloroplast genes rbcL and psaB, we demonstrate the existence of three cryptic species that were previously confused under Prasiola crispa (Lightfoot) Kützing. Genuine P. crispa occurs in Antarctica; its presence was confirmed by comparison with the rbcL sequence of the type specimen (from the Isle of Skye, Scotland). Prasiola antarctica Kützing is resurrected as an independent species to designate algae with gross morphology identical to P. crispa but robustly placed in a separate lineage. The third species is represented by specimens identified as P. calophylla (Carmichael ex Greville) Kützing in previous studies, but clearly separated from European P. calophylla (type locality: Argyll, Scotland); this alga is described as P. glacialis sp. nov. The molecular data demonstrated the presence of P. crispa in Maritime and Continental Antarctica. P. antarctica was recorded from the Antarctic Peninsula and Shetland Islands, and P. glacialis from the Southern Ocean islands and coast. Such unexpected cryptic diversity highlights the need for a taxonomic reassessment of many published Antarctic records of P. crispa. The results also indicate that marine species of Prasiola form a well-supported monophyletic group, whereas the phylogenetic diversity of freshwater species is higher than previously suspected (at least three separate lineages within the genus include species living in this type of environments).

First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh.

Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supply's pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 µg g⁻¹ dry weight in the lake sample and 119.4 µg g⁻¹ dry weight or 1.3 pg cell⁻¹ in UCFS10. This is the first confirmation of a saxitoxin-producing species in New Zealand and the first report of saxitoxin production by a species of Scytonema.

Phylogeny and taxonomy of Xanthophyceae (Stramenopiles, Chromalveolata).

The stramenopile class Xanthophyceae has been variously divided into two to seven orders. Previous molecular phylogenetic analyses of the class have had limited taxon/gene sampling or have focused on particular taxa. Despite these limitations para-polyphyletic groups have been identified at different taxonomic ranks. To investigate the phylogeny of the Xanthophyceae, a multiple alignment containing SSU rDNA, rbcL and psaA gene portions was analyzed according to bayesian inference, maximum likelihood, minimum evolution and maximum parsimony methods. This multigenic approach allowed robust resolution of evolutionary relationships within Xanthophyceae and the proposal of a taxonomic revision within the class. The best statistically supported tree either showed/or confirmed that several taxa at different taxonomic levels were para-polyphyletic. Ten epitypes were formally proposed for species of Chlorellidium, Botrydiopsis and Bumilleriopsis. Boundaries of these coccoid genera were redefined as monophyletic groups. Four major clades received strong statistical support. These accommodated most of the studied coccoid, filamentous and siphonous Xanthophyceae. Unicellular flagellates, amoeboid and palmelloid taxa were not included in the study. Botrydiopsis pyrenoidosa was excluded from Xanthophyceae and placed incertae sedis.

Molecular phylogeny and evolution of the order Tribonematales (Heterokonta, Xanthophyceae) based on analysis of plastidial genes rbcL and psaA.

Tribonematales is an order of filamentous algae in the class Xanthophyceae (Heterokonta). Few molecular studies, all with a limited taxon sampling, have previously investigated its evolutionary history and phylogenetic relationships. We sequenced the chloroplast-encoded rbcL and psaA genes of several tribonematalean species and of several coccoid and siphonous forms that previous studies revealed to be strictly related to Tribonematales. Multiple alignments included mostly new sequences obtained from 42 taxa. Phylogenetic reconstructions were performed using the maximum likelihood method. The rbcL and psaA data sets were analyzed independently and combined in a single multiple alignment. Neither rbcL nor psaA genes showed intraspecific sequence variation. The former proved to be a better diagnostic marker than the latter for characterization of species. We explored effects produced on phylogenetic outcomes by selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on the combined data set. There is strong statistical support for trees that show several currently recognized taxonomic groups to be polyphyletic. The siphonous orders Botrydiales and Vaucheriales do not form a clade. Botrydiales and Tribonematales are polyphyletic as are the families Botrydiaceae, Centritractaceae and Tribonemataceae and the genera Xanthonema and Bumilleriopsis. We tentatively define new boundaries of the Tribonematales to include both coccoid and filamentous species having a bipartite cell wall and also the siphonous members of the genus Botrydium. Also, our results support morphological convergence at all taxonomic ranks in the evolution of the Xanthophyceae.

Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes.

Xanthophyceae are a group of heterokontophyte algae. Few molecular studies have investigated the evolutionary history and phylogenetic relationships of this class. We sequenced the nuclear-encoded SSU rDNA and chloroplast-encoded rbcL genes of several xanthophycean species from different orders, families, and genera. Neither SSU rDNA nor rbcL genes show intraspecific sequence variation and are good diagnostic markers for characterization of problematic species. New sequences, combined with those previously available, were used to create different multiple alignments. Analyses included sequences from 26 species of Xanthophyceae plus three Phaeothamniophyceae and two Phaeophyceae taxa used as outgroups. Phylogenetic analyses were performed according to Bayesian inference, maximum likelihood, and maximum parsimony methods. We explored effects produced on the phylogenetic outcomes by both taxon sampling as well as selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on a data set including both SSU rDNA and rbcL sequences. Trees obtained in this study show that several currently recognized xanthophycean taxa do not form monophyletic groups. The order Mischococcales is paraphyletic, while Tribonematales and Botrydiales are polyphyletic even if evidence for the second order is not conclusive. Botrydiales and Vaucheriales, both including siphonous taxa, do not form a clade. The families Botrydiopsidaceae, Botryochloridaceae, and Pleurochloridaceae as well as the genera Botrydiopsis and Chlorellidium are polyphyletic. The Centritractaceae and the genus Bumilleriopsis also appear to be polyphyletic but their monophyly cannot be completely rejected with current evidence. Our results support morphological convergence at any taxonomic rank in the evolution of the Xanthophyceae. Finally, our phylogenetic analyses exclude an origin of the Xanthophyceae from a Vaucheria-like ancestor and favor a single early origin of the coccoid cell form.