Another origin of coloniality in volvocaleans: the phylogenetic position of Pyrobotrys arnoldi (Spondylomoraceae, Volvocales).

Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a-apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic "Pascherina tetras" SAG 159-1 was also analyzed.

Feasibility of removing surface deposits on stone using biological and chemical remediation methods.

The study was conducted on alterations found on stone artwork and integrates microbial control and a biotechnological method for the removal of undesirable chemical substances. The Demetra and Cronos sculptures are two of 12 stone statues decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). An initial inspection of the statues revealed putative black crusts and highlighted the microbial contamination causing discoloration. In 2006, the Cultural Heritage Superintendence of Trento commissioned us to study and remove these chemical and biological stains. Stereomicroscopy characterised the stone of the sculptures as oolitic limestone, and infrared analyses confirmed the presence of black crusts. To remove the black crusts, we applied a remediation treatment of sulphate-reducing bacteria, which removes the chemical alteration but preserves the original stone and the patina noble. Using traditional and biomolecular methods, we studied the putative microbial contamination and confirmed the presence of biodeteriogens and chose biocide Biotin N for the removal of the agents causing the discolouration. Denaturing gradient gel electrophoresis fluorescent in situ hybridisation established that Cyanobacteria and green algae genera were responsible for the green staining whereas the black microbial contamination was due to dematiaceous fungi. After the biocide Biotin N treatment, we applied molecular methods and demonstrated that the Cyanobacteria, and most of the green algae and dematiaceous fungi, had been efficiently removed. The reported case study reveals that conservators can benefit from an integrated biotechnological approach aimed at the biocleaning of chemical alterations and the abatement of biodeteriogens.

Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: an overview.

The presence and deteriorating action of micro-organisms on monuments and stone works of art have received considerable attention in the last few years. Knowledge of the microbial populations living on stone materials is the starting point for successful conservation treatment and control. This paper reviews the literature on cyanobacteria and chlorophyta that cause deterioration of stone cultural heritage (outdoor monuments and stone works of art) in European countries of the Mediterranean Basin. Some 45 case studies from 32 scientific papers published between 1976 and 2009 were analysed. Six lithotypes were considered: marble, limestone, travertine, dolomite, sandstone and granite. A wide range of stone monuments in the Mediterranean Basin support considerable colonization of cyanobacteria and chlorophyta, showing notable biodiversity. About 172 taxa have been described by different authors, including 37 genera of cyanobacteria and 48 genera of chlorophyta. The most widespread and commonly reported taxa on the stone cultural heritage in the Mediterranean Basin are, among cyanobacteria, Gloeocapsa, Phormidium and Chroococcus and, among chlorophyta, Chlorella, Stichococcus and Chlorococcum. The results suggest that cyanobacteria and chlorophyta colonize a wide variety of substrata and that this is related primarily to the physical characteristics of the stone surface, microclimate and environmental conditions and secondarily to the lithotype.

Identification of freshwater Phycodnaviridae and their potential phytoplankton hosts, using DNA pol sequence fragments and a genetic-distance analysis.

Viruses that infect phytoplankton are an important component of aquatic ecosystems, yet in lakes they remain largely unstudied. In order to investigate viruses (Phycodnaviridae) infecting eukaryotic phytoplankton in lakes and to estimate the number of potential host species, samples were collected from four lakes at the Experimental Lakes Area in Ontario, Canada, during the ice-free period (mid-May to mid-October) of 2004. From each lake, Phycodnaviridae DNA polymerase (pol) gene fragments were amplified using algal-virus-specific primers and separated by denaturing gradient gel electrophoresis; 20 bands were extracted from the gels and sequenced. Phylogenetic analysis indicated that freshwater environmental phycodnavirus sequences belong to distinct phylogenetic groups. An analysis of the genetic distances "within" and "between" monophyletic groups of phycodnavirus isolates indicated that DNA pol sequences that differed by more than 7% at the inferred amino acid level were from viruses that infect different host species. Application of this threshold to phylogenies of environmental sequences indicated that the DNA pol sequences from these lakes came from viruses that infect at least nine different phytoplankton species. A multivariate statistical analysis suggested that potential freshwater hosts included Mallomonas sp., Monoraphidium sp., and Cyclotella sp. This approach should help to unravel the relationships between viruses in the environment and the phytoplankton hosts they infect.

New design strategy for development of specific primer sets for PCR-based detection of Chlorophyceae and Bacillariophyceae in environmental samples.

Studying aquatic microalgae is essential for monitoring biodiversity and water quality. We designed new sets of 18S rRNA PCR primers for Chlorophyceae and Bacillariophyceae by using the ARB software and implementing a virtual PCR program. The results of specificity analysis showed that most of the targeted algal families were identified and nontargeted organisms, such as fungi or ciliates, were excluded. These newly developed PCR primer sets were also able to amplify microalgal rRNA genes from environmental samples with accurate specificity. These tools could be of great interest for studying freshwater microalgal ecology and for developing bioindicators of the health status of aquatic environments.

Phylogenetic and molecular analysis of hydrogen-producing green algae.

A select set of microalgae are reported to be able to catalyse photobiological H(2) production from water. Based on the model organism Chlamydomonas reinhardtii, a method was developed for the screening of naturally occurring H(2)-producing microalgae. By purging algal cultures with N(2) in the dark and subsequent illumination, it is possible to rapidly induce photobiological H(2) evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. This procedure can be used to test algal species existing as axenic or mixed cultures for their ability to produce H(2). Using this system, five algal isolates capable of H(2) production were identified in various aquatic systems. A phylogenetic tree was constructed using ribosomal sequence data of green unicellular algae to determine if there were taxonomic patterns of H(2) production. H(2)-producing algal species were seen to be dispersed amongst most clades, indicating an H(2)-producing capacity preceded evolution of the phylum Chlorophyta.

New subgroup of Bacteroidetes and diverse microorganisms in Tibetan plateau glacial ice provide a biological record of environmental conditions.

We recovered microorganisms from five ice core samples from three glaciers (Puruogangri, Malan, and Dunde) located in the Tibetan Plateau in China and analyzed their small subunit rRNA gene sequences. Most of the bacterial sequences were unknown previously; the most closely related known sequences were from bacteria of the Proteobacteria, Bacteroidetes, and Actinobacteria phyla. Chlorophyta, Streptophyta, Ciliophora, and fungal groups were represented among the 18S rRNA gene sequences that we obtained. The most abundantly represented glacial bacteria were Bacteroidetes, and Chlamydomonas was the predominant eukaryote. Comparative analysis showed that the Bacteroidetes sequences obtained from this study were highly similar to one another but most were only distantly related to previously characterized Bacteroidetes (<92% identity). We propose that our Bacteroidetes sequences represent two novel subgroups: one at the family level and one at the genus level. The unique ice environment and the high abundance of Bacteroidetes, combined with the coexistence of a high abundance of psychrophilic Chlamydomonas, strongly suggests that there is a viable ecosystem on the surface of Tibetan glaciers. Comparisons of microbial community structures in the five ice samples showed distinct differences, likely due to environmental differences in the locations in which the samples were obtained.

Coccomyxa sp. (chlorophyta: chlorococcales), a new pathogen in mussels (Mytilus galloprovincialis) of Vigo estuary (Galicia, NW Spain).

In this work, we describe the occurrence of irregular shaped green aggregations in the mantle, gill filaments, adductor muscle, visceral mass and haemolymph of wild mussels (Mytilus galloprovincialis) collected from the Vigo estuary (Galicia, NW Spain). Microscopic examination of these masses revealed that they consist of intracellular green algae which are spherical to oval in shape, 5 microm in length and 3 microm in width, without flagella and with a smooth surface. The algal cells present a small single nucleus, a mitochondrion, 1-2 parietal chloroplasts and lack pyrenoids. Reproduction is by formation of 2-4 autospores or daughter cells. Pigment analysis reveals the presence of photopigments typical of green algae in addition to alloxanthin, diadinoxanthin and diatoxanthin. These carotenoids are noted for the first time in a parasitic chlorophyte. The signs of infection, together with the morphological observations, suggest that this parasitic algae may be Coccomyxa parasitica. However, further molecular studies are required for confirmation. This is the first report of Coccomyxa algae parasitizing the species M. galloprovincialis.

The carotenogenesis pathway via the isoprenoid-beta-carotene interference approach in a new strain of Dunaliella salina isolated from Baja California Mexico.

D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.

Characterization of Desmodesmus pleiomorphus isolated from a heavy metal-contaminated site: biosorption of zinc.

Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn(2+) concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn(2+) uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.

Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol.

The purpose of the present work was to study the potential biotechnological use of Dunaliella species isolated from a hypersaline lake in Turkey. Dunaliella spp. grown in Johnson's medium were isolated and their glycerol production was studied in a batch system in order to determine the optimal conditions required for the highest glycerol accumulation. In the experiments performed with four newly isolated Dunaliella spp., the maximum glycerol accumulation was obtained at 20% NaCl concentration, and pH 6 (for strains T1 and T2) and pH 9 (for strains T3 and T4). Biomass production by strain T2 was significantly higher that by the other strains but the highest glycerol production in broth was obtained by strain T1 followed by strain T2. Strain T1 showed high glycerol production, i.e. 452.57microg/ml of culture broth at 20% NaCl concentration. The highest glycerol accumulation on both dry weight and cell basis was obtained with strain T1, followed by strains T3 and T4 (55.01, 50.16, and 40.23microg/10(6) cells (or pg/cell), respectively) at 25% NaCl concentration. When the high initial inoculum concentration was used at 25% NaCl concentration, strain T1 had the shortest (approximately 10-15days) lag period. This study shows that the isolated strains T1 and T2 can be used for glycerol production because of their high productivity.

Unusual medium-chain polyunsaturated fatty acids from the snow alga Chloromonas brevispina.

The gas chromatography-mass spectrometry (GC-MS) method was used to identify unusual medium-chain polyunsaturated fatty acids (PUFAs) in the snow alga Chloromonas brevispina collected in 2006 from surface layers of a snow field with conspicuous green patches in Bohemian Forest (Czech Republic). PUFAs formed more than 75% total fatty acids. Among them, mass spectroscopy of picolinyl esters showed sizable proportions of medium-chain PUFA, e.g., 5,8,11-tetradecatrienoic and 6,9,12-pentadecatrienoic acids. The high relative content of PUFA indicates that PUFA are an important element ensuring cell survival. Our report appears to be the first to describe the presence of short- and medium-chain PUFAs in green psychrophilic algae of the genus Chloromonas.

Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea.

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately 16-24 microm long and 10-15 microm wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of beta-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of beta-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate beta- carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.

Performance evaluation of a simple wastewater treatment system comprised by UASB reactor, shallow polishing ponds and coarse rock filter.

The work investigates a small full-scale wastewater treatment system comprised by the following units in series: UASB reactor, three polishing ponds and one coarse rock filter. The overall performance of the system is analyzed based on three years of monitoring using physical-chemical and biological parameters. Good organic matter, suspended solids and ammonia removal is achieved, together with excellent coliform removal (5.70 log units). Mean effluent concentrations of the main parameters are: BOD: 39 mg/L; COD: 109 mg/L; SS = 41 mg/L; ammonia: 10 mg/L; E. coli: 540 MPN/100 mL, indicating compliance with many regulations for effluent discharge and reuse. Main algal classes found in the ponds and final effluent were chlorophyta and euglenophyta. The system is completely unmechanized and has a relatively small total hydraulic retention time (less than 13 days), compared with most natural treatment processes. No sludge removal from the ponds and filter has been necessary so far.

Microbiota within the perennial ice cover of Lake Vida, Antarctica.

Lake Vida, located in the McMurdo Dry Valleys, Antarctica, is an 'ice-sealed' lake with approximately 19 m of ice covering a highly saline water column (approximately 245 ppt). The lower portions of the ice cover and the lake beneath have been isolated from the atmosphere and land for circa 2800 years. Analysis of microbial assemblages within the perennial ice cover of the lake revealed a diverse array of bacteria and eukarya. Bacterial and eukaryal denaturing gradient gel electrophoresis phylotype profile similarities were low (<59%) between all of the depths compared (five depths spanning 11 m of the ice cover), with the greatest differences occurring between surface and deep ice. The majority of bacterial 16S rRNA gene sequences in the surface ice were related to Actinobacteria (42%) while Gammaproteobacteria (52%) dominated the deep ice community. Comparisons of assemblage composition suggest differences in ice habitability and organismal origin in the upper and lower portions of ice cover. Specifically, the upper ice cover microbiota likely reflect the modern day transport and colonization of biota from the terrestrial landscape, whereas assemblages in the deeper ice are more likely to be persistent remnant biota that originated from the ancient liquid water column of the lake that froze.

Agricultural fertilizers as economical alternative for cultivation of Haematococcus pluvialis.

A Haematococcus pluvialis strain isolated from the ruins of Ephesus in Turkey was investigated as regards its adaptation to laboratory conditions and maximum growth rate. In the first stage of the experiment, the growth of H. pluvialis was compared in common culture media. Furthermore, in an effort to minimize the culture costs, the second stage of the experiment compared the growth rate in the culture medium selected in the first stage with that in commercial plant fertilizers. The results demonstrated that the maximum cell concentration of 0.90 g/l, corresponding to a growth rate of 0.150 d(-1), was found with an N-P-K 20:20:20 fertilizer under a light intensity of 75 micromol photons m(-2) s(-1) on the 12th day of cultivation.

Haematococcus pluvialis cell-mass sensing using ultraviolet fluorescence spectroscopy.

A simple whole-cell-based sensing system is proposed for determining the cell mass of H. pluvialis using ultraviolet fluorescence spectroscopy. An emission signal at 368 nm was used to detect the various kinds of green, green-brown, brown-red, and red H. pluvialis cells. The fluorescence emission intensities of the cells were highest at 368 nm with an excitation wavelength of 227 nm. An excitation wavelength of 227 nm was then selected for cell-mass sensing, as the emission fluorescence intensities of the cell suspensions were highest at this wavelength after subtracting the background interference. The emission fluorescence intensities of HPLC-grade water, filtered water, and HPLC-grade water containing a modified Bold's basal medium (MBBM) were measured and the difference was less than 1.6 for the selected wavelengths. Moreover, there was no difference in the emission intensity at 368 nm among suspensions of the various morphological states of the cells. A calibration curve of the fluorescence emission intensities and cell mass was obtained with a high correlation (R(2)=0.9938) for the various morphological forms of H. pluvialis. Accordingly, the proposed method showed no significant dependency on the various morphological cell forms, making it applicable for cell-mass measurement. A high correlation was found between the fluorescence emission intensities and the dry cell weight with a mixture of green, green-brown, brown-red, and red cells. In conclusion, the proposed model can be directly used for cell-mass sensing without any pretreatment and has potential use as a noninvasive method for the online determination of algal biomass.

Involvement of zeaxanthin and of the Cbr protein in the repair of photosystem II from photoinhibition in the green alga Dunaliella salina.

A light-sensitive and chlorophyll (Chl)-deficient mutant of the green alga Dunaliella salina (dcd1) showed an amplified response to irradiance stress compared to the wild-type. The mutant was yellow-green under low light (100 micromol photons m(-2) s(-1)) and yellow under high irradiance (2000 micromol photons m(-2) s(-1)). The mutant had lower levels of Chl, lower levels of light harvesting complex II, and a smaller Chl antenna size. The mutant contained proportionately greater amounts of photodamaged photosystem (PS) II reaction centers in its thylakoid membranes, suggesting a greater susceptibility to photoinhibition. This phenotype was more pronounced under high than low irradiance. The Cbr protein, known to accumulate when D. salina is exposed to irradiance stress, was pronouncedly expressed in the mutant even under low irradiance. This positively correlated with a higher zeaxanthin content in the mutant. Cbr protein accumulation, xanthophyll cycle de-epoxidation state, and fraction of photodamaged PSII reaction centers in the thylakoid membrane showed a linear dependence on the chloroplast 'photoinhibition index', suggesting a cause-and-effect relationship between photoinhibition, Cbr protein accumulation and xanthophyll cycle de-epoxidation state. These results raised the possibility of zeaxanthin and Cbr involvement in the PSII repair process through photoprotection of the partially disassembled, and presumably vulnerable, PSII core complexes from potentially irreversible photooxidative bleaching.

Marsh stars in Liverpool.

INTRODUCTION/METHODS: After the recognition of contaminating algae on histopathological sections stained by periodic acid Schiff (PAS) and Grocott methods, a detailed audit was undertaken to assess the extent of contamination and its possible source. RESULTS: The contaminating organism was a member of the staurastrum genus of Chlorophyta, star shaped organisms commonly found in fresh water marshes. The organisms were seen on sections stained by the diastase-PAS, PAS, or Grocott methods and on cytological preparations between July 2003 and May 2004. It is unlikely that contamination of water baths or concentrated staining solutions was to blame, and a more general contamination of the laboratory water supply is the most likely source. CONCLUSIONS: Contaminating organisms may appear on histological and cytological material and their nature and source should be investigated. Although in this instance, confusion with pathologically important organisms was minimal, algae may occasionally cause significant disease.

A fungus-type beta-galactofuranan in the cultivated Trebouxia photobiont of the lichen Ramalina gracilis.

A structural characterization of polysaccharides extracted from the aposymbiotically cultured photobiont of the lichen Ramalina gracilis was carried out in order to compare them with those previously found in the symbiotic thallus. The photobiont was isolated from thallus fragments, following the method of Yamamoto, and cultivated in a liquid nutrient medium. Freeze-dried cells were defatted, and the polysaccharides extracted successively with water and aq. 10% KOH, each at 100 degrees C. After purification, the soluble fractions provided a polysaccharide containing a (1-->5)-linked beta-galactofuranosyl backbone, substituted in a small proportion at O-6 by beta-Galf units. Amylose was also found, as insoluble material obtained on freeze-thawing of the alkaline extract. These polysaccharides have not been found in the symbiotic thallus of Ramalina gracilis, which contained only water-soluble (isolichenan) and insoluble glucans (nigeran and laminaran), and galactomannan. Surprisingly, the galactofuranan has similarities with those found in some fungal cell walls.