The extensive diversity of microalgae provides an opportunity to undertake bioprospecting for species possessing features suited to commercial scale cultivation. The outdoor cultivation of microalgae is subject to extreme temperature fluctuations; temperature tolerant microalgae would help mitigate this problem. The waters of the Roman Baths, which have a temperature range between 39°C and 46°C, were sampled for microalgae. A total of 3 green algae, 1 diatom and 4 cyanobacterial species were successfully isolated into 'unialgal' culture. Four isolates were filamentous, which could prove advantageous for low energy dewatering of cultures using filtration.
Lipid content, profiles and growth rates of the isolates were examined at temperatures of 20, 30, 40°C, with and without nitrogen starvation and compared against the oil producing green algal species, Chlorella emersonii. Some isolates synthesized high levels of lipids, however, all were most productive at temperatures lower than those of the Roman Baths. The eukaryotic algae accumulated a range of saturated and polyunsaturated FAMEs and all isolates generally showed higher lipid accumulation under nitrogen deficient conditions (Klebsormidium sp. increasing from 1.9% to 16.0% and Hantzschia sp. from 31.9 to 40.5%). The cyanobacteria typically accumulated a narrower range of FAMEs that were mostly saturated, but were capable of accumulating a larger quantity of lipid as a proportion of dry weight (M. laminosus, 37.8% fully saturated FAMEs). The maximum productivity of all the isolates was not determined in the current work and will require further effort to optimise key variables such as light intensity and media composition.
'The transport fuel sector constitutes a large proportion of global energy demand but renewable alternatives, in comparison to other energy sectors, are largely underdeveloped (Schenk et al. 2008). To be competitive renewable liquid fuels must match the performance, energy density and versatility of current fossil fuels (Rupprecht 2009). It is therefore imperative that renewable, energy dense liquid fuels capable of integration into the existing infrastructure are rapidly developed.
One renewable liquid fuel is biodiesel, comprised of the fatty acid methyl esters (FAMEs) produced from the esterification of biologically derived oils. Biodiesel contains chain lengths between C14-C24 with varying degrees of unsaturation (Varfolomeev and Wasserman, 2011). The FAME profile is also dependent on the specific producing organism as well as its growing conditions (Saraf and Thomas, 2007). Thus, biodiesel tends to have variable fuel properties that can substantially affect engine performance (Fortman et al. 2008). Biodiesel contains a relatively high oxygen content by weight which results in more complete combustion than mineral diesel resulting in lower CO, particulate matter and hydrocarbon emissions (Song et al., 2008). Currently biodiesel is produced from agricultural crops such as rapeseed, soybean or palm that occupy limited agricultural resources, are produced on a seasonal basis and in most cases generate a low energy return (Rittmann, 2008). They also pose a threat to biodiversity, as marginal land is often ecologically valuable, constituting habitat or acting as a watershed (Gressel, 2008).
In an attempt to replace these first generation feedstocks, there has been a resurgence of interest in developing oleaginous microalgae. Algal biofuels have potentially high energy returns and a small ecological footprint (Groom et al. 2008), though the FAME profile is reportedly more variable than those produced by terrestrial crops (Haik et al. 2011). Microalgae have the potential for all year round harvest (Chisti, 2008), are lipid-rich and offer the prospect of harnessing the residual biomass in conventional biomass technologies (Um and Kim, 2009). Algae culture can be coupled to industrial CO2 sequestration (Chisti, 2007), bioremediation or scrubbing of waste streams (Pittman et al. 2011). In addition, algae are capable of producing a multitude of other high-value products including biopolymers, vitamins, antioxidants, polysaccharides, proteins, and pharmaceuticals (Raja et al., 2008). Economic analysis demonstrates that co-production of these products is currently essential for commercial viability (Ono and Cuello, 2006).
Algae are responsible for 50% of global CO2 fixation and have colonised diverse ecological habitats from oceans to hot springs, snowfields and waste streams (Croft et al. 2006). Microalgae are best described as unicellular, microscopic (2-200μm) photosynthetic organisms as they have both prokaryotic (cyanobacteria) and eukaryotic representatives, with over 300,000 species (Pulz and Gross, 2004). The most abundant microalgae in any given environment are generally the diatoms, green algae, cyanobacteria and golden algae (Mutanda et al. 2011).
Microalgae which can accumulate lipids at over 20% of their dry weight are referred to as oleaginous species, most of which belong to the green algae and diatoms (Pulz and Gross, 2004). Cyanobacteria are also capable of accumulating lipid, yet are comparatively understudied with regard to lipid content and profile (Karatay and Dönmez 2011). Chlorella spp. is a commonly studied oleaginous genera with a very high CO2 fixation rate and lipid content compared to other green algae. In the long term, genetic modification might well have the biggest impact on realising fuels from microalgae but algal bioengineering is in its infancy (Chisti and Yan, 2011; Purton and Stevens, 1997). In addition, transgenic cells usually exhibit lower fitness as well as there being restrictions and public concern over their commercial use, therefore, genetic modification must 'compliment and not substitute screening of new species' (Pulz and Gross, 2004).
Since microalgae are a diverse group of organisms, with large variations in growth requirements, growth rate, fatty acid content and the ability to produce other valuable products, the screening and isolation of new algal species is an important goal in developing algal biofuels (Hu et al. 2008). Choice of species is an essential consideration for any applied microalgal biotechnology (Ratha and Prasanna, 2012). The dilute nature of microalgal cultures means extensive dewatering is required prior to biomass processing, which significantly raises energy costs using current methods such as centrifugation, electrophoresis and floatation (Chisti and Yan, 2011). Harvesting costs are therefore an important consideration when screening for new strains, as differences in morphology can have a substantial impact on the cost of production (Mutanda et al. 2011). Whilst low energy methods such as filtration are challenging with unicellular microalgal species these can be very effective for filamentous species (Uduman et al. 2010 and Mohan et al. 2010).
Bioprospecting of diverse aquatic environments offers a way to obtain new oleaginous microalgae with features suited to commercialisation. Hypersaline and thermophillic springs have proved ideal environments for the identification novel microalgae (Mutanda et al. 2011). Extremophilic algae may be beneficial for industrial applications, as the unique environment minimizes contamination risk, particularly in open pond systems, and can provide buffering against fluctuations in temperature (Pulz and Gross, 2004). Significantly, the most productive commercially cultivated species could be considered extremophillic (Xu et al. 2009).
Commonly cultured microalgal species have optimal growth between 16-27°C, with temperatures below 16°C resulting in slow growth, and temperatures above 35°C often being lethal (Barsanti and Gualtieri, 2006). Although thermotolerant microalgal species have been isolated from hot springs (primarily in the USA and Japan), few have been extensively studied to date (Ratha and Prasanna, 2012).
The largest geothermal spring in the UK is situated in the city of Bath at the Roman Baths (ST750647) (Atkinson and Davison, 2002). A series of 3 thermal springs first supply water at a rate 720 l h-1 to the Kings Bath (46.5°C), which then drains into the Great Bath 39.0°C; (Andrews et al. 1982). The baths were gradually constructed by Roman settlers between 70-370 AD, but fell into disrepair gradually becoming buried during the 5th century as the Romans withdrew from Britain (pers. comm. Tom Byrne). Excavation of the Baths began in 1878 and have remained open to the elements for the last 130 years (Kellaway, 1991). Since thermotolerance is attractive for biofuel production we set out to bioprospect the hot waters of the Roman Baths for microalgal strains with an ability to produce lipids for biodiesel production.
Samples of water (1l) were taken from the Great Bath and the Kings Bath and analysed by Severn Trent Services, the pH and temperature were measured on site. Three visible filamentous algae were sampled from scrapings taken from microbial mats submerged beneath the water surface. Approximately 90 l of water from the Great Bath was filtered through a custom-made filter-stack at 3 positions around the Great Bath (the water entry point, exit point and opposite). The custom filter stack comprised of an acrylic tube with a coarse 3 mm mesh metal filter, a sponge filter and a Whatman GF fine filter paper. The GF filter paper layer was used as a sample for further culturing.
The algae loaded GF filter papers were immediately cultured using a method adapted from Ferris and Hirsch (1991) ('filter paper method'). Filters were placed on top of a stack of 5 Whatman GF papers saturated with a mixture of 1l BBM:BG media 1:1, inside a glass petridish with lid, before sealing with Parafilm. Samples were then left at room temperature and in low light (on lab bench) until colonies appeared.
Small samples were taken from colonies and placed on a microscope slide with a coverslip for quick examination under microscope Nikon Eclipse 90i and confocal D-Eclipse. Colonies with green or blue-green pigmentation (either single celled, filamentous or of diatom morphology) were picked off, diluted and re-plated on filter paper as described above, as well as on BBM pH 6.5 and BG-11 pH 7.1 agar plates (1% agar). Where possible, both filter paper and agar methods were repeated until a unialgal culture was derived from single colonies comprised of one alga (assessed under inverted microscope Nikon eclipse TE2000-S).
The method of (Vaara et al. 1979) was used ('agar plate scoring method') to clean filamentous samples of contaminant fungal species. Agar plates were scored perpendicular to the end of inoculation. Plates were covered in black cloth with the end opposite the inoculation site exposed to a light source. This arrangement encourages growth of algal filaments along the agar scores, towards the light, thereby shedding fungal and bacterial contaminants.
All isolates were deposited in the CCAP culture collection and have been assigned the following collection numbers. Oscillatoria sancta (CCAP 1459/46), Microcoleus chthonoplastes (CCAP 1449/2), Mastigocladus laminosus (CCAP 1447/3), Klebsormidium sp. (CCAP 335/20), Coelastrella saipanensis (CCAP 217/9), Chroococcidiopsis thermalis (CCAP 1423/1) and Hantzschia sp. (CCAP 1030/1).
An experiment to determine a suitable temperature range for further analysis was carried out using heat blocks. All isolates together with a reference alga, Chlorella emersonii (Ce), were cultivated in triplicate in 15 ml Sterilin falcon tubes at temperatures between 25-60°C at intervals of 5°C. Tubes were shaken manually twice a day and the lids opened once a day to allow for gas exchange. The growth was assessed visually by comparing the tubes across the temperature ranges. As a result of the experiment 20°C, 30°C and 40°C were chosen for further experiments.
Each species was cultivated in 2 × 250ml conical flasks with 100 ml of suitable media: Bolds Basal Medium pH 6.5 (BBM) for eukaryotes and Blue Green-11 Medium pH 7.1 (BG-11) for prokaryotes and a 'Diatom Media' for the diatom (DM), comprised of BBM with the addition of 100μl B vitamins (0.0024% B1B7B12 1:1:1) and 100μl sodium silicate. The flasks were inoculated with 100μl stock cultures. Cultures were incubated in a plant growth chamber (in Sanyo MLR-351) on a rotary platform (Sanyo MIR-S100) at 100 rpm, with a 100-150 μmol m-2 s-1 of light on a 12 hour light/dark cycle. Chroococcidiopsis thermalis and Hantzschia sp. culture flasks were covered in a double layer of neutral density filter (StageElectrics, Bristol, UK) due to their sensitivity to light.
After 12 days one flask of each culture was pelleted and re-suspended in an appropriate nitrogen free media and cultivated for a further 6 days. Samples were then pelleted by centrifugation and lyophilised.
For single celled algae, growth measurements were recorded as cell counts using a haemocytometer and dry weights calculated by lyophilising 2 ml of the culture. Filamentous algae had a tendency to form mats or aggregates in the flasks and to stick to the walls of the vessel. Consequently, growth measurements for filamentous algae were done by initiating cultures in pre-weighed 15 ml falcon tubes containing 7.5 ml media. The algae were continuously mixed using a bloodtube rotator (Stuart Scientific SB1), Stone, Staffordshire, UK. At 3 day intervals 3 tubes were removed, centrifuged to remove water and lyophilised.
Algal cultures were grown in 100 ml of appropriate liquid media under culture conditions described above. DNA extracted from 50 ml of each culture was centrifuged and the wet pellet transferred to a 1.5 ml Eppendorf. 300 μl of 20 mM Na-phosphate buffer (pH 8), 150 μl of lysis solution (10% sodium dodecyl sulphate, 0.5M Tris HCl (pH 8), 0.1M NaCl) and sufficient 0.1 mm silica/zirconium beads added to make a paste which was then ground for 5mins on ice using a bench drill fitted with an Eppendorf micropestle. 300 μl DNA extraction buffer was added and centrifuged 13,000 rpm for 1 min; the supernatant was transferred to a fresh 1.5 ml Eppendorf. This process was then repeated. An equal volume of phenol-chloroform-isoamylalcohol (25:24:1) was added, mixed well and centrifuged for 3 min at 10,000 xg. The upper aqueous layer was then transferred to a fresh 1.5 ml Eppendorf. The phenol-chloroform-isoamylalcohol extraction repeated. DNA was precipitated with an equal volume of 70% ethanol and the pellet washed with 3 rounds of 70% ethanol followed by centrifugation (13,500 xg, 5 mins). Finally, the ethanol was removed and the pellet dried at room temperature for Y-X mins before resuspending in 50 μl of sterile deionised H2O.
0.5 μl of DNA was added to a 200 μl thin walled PCRtube containing 12.5 μl DreamTaq Green Master Mix (Fermentas, UK), 1 μl forward primer and 1 μl reverse primers (Additional file 1: Table S1), 10 μl DNase-free water. Cycling conditions were 95°C for 5 min followed by 32 cycles (95°C 45s, X°C 45s, 72°C 120s (where X is the annealing temperature for each primer, indicated in (Additional file 1: Table S1)) with a final cycle of 72°C for 10 min. DNA sequences were checked manually, corrected and assembled using Sequencher 4.10.1 (Gene Codes Cooporation, Ann Arbor, Michigan, USA), sequences were assembled using corrected sequences were used to interrogate the NCBI online database using BLAST. The attribution of genus and/or species identity to the isolates was based on the BLAST total score. Total score is not included in the results (Table 1) as it summarises and compares all data from resultant matches for a single query (arbitrary value for each sequence analysed). Instead, Table 1 contains 'query coverage' and 'max ident' which better describe the 'quality' of each final match. In order to be consistent 'total score' from the BLAST outputs was used as a means of identification. Where scores were ambiguous, decisions were based on morphology at the light-microscopy level.
Sequences compared to the NCBI online database using BLAST. Matches based on 'Max Score'. Date accessed 17.01.2012.
*% of sequence covered by database hit **% similarity of sequence covered by hit ***The score is calculated from the sum of the match rewards and the mismatch (independently for each segment).
H. sp. was identified based on morphology, using images taken from a slide preparation outlined below. An obtained pellet of diatom cells was washed with deionised water twice and centrifuged at 3000 rpm over 10 mins. Supernantant was removed and pellet resuspended in 1ml 30% (w/v) hydrogen peroxide and mixed well. Tube then placed in a water bath at 80-90°C for 1 hr and then allowed to cool. Sample was transferred to 15 ml falcon tube with ~10 ml deionised H2O, mixed and centrifuged at 3000 rpm for 3 min. Supernatant then removed and pellet washed twice with distilled water, centrifuging each time and removing the supernatant. Working in a clean flow hood 0.5 ml of mixed suspension was dropped onto coverslips and dried using a hotplate at ~50°C. 1 drop of DPX mounting medium was dropped onto glass slides and using forceps the coverslips with dried diatom were inverted and placed over the drops of DPX. Assembled slides were then dried on the hotplate (at 80°C) overnight. Images were assessed by Dr. S. Spaulding (Colorado State University).
NCBI accession numbers for the Roman Bath isolates are as follows; Coelastrella saipanensis JX316760, Klebsormidium sp. JX316761, Hantzschia sp. JX316762, Chroococcidiopsis thermalis JX316763 and Masticocladus laminosus JX316764. For the purposes of this paper isolates Os and Mc have been identified as Oscillatoria sancta and Microcoleus chthonoplastes. However both sequences contained alignment gaps not acceptable to the NCBI database. Both isolates require re-sequencing for more accurate species identification.
100 μl of cell culture was added to 25 μl DMSO and 1 μl 1% nile red dye in acetone in an eppendorf, mixed well and allowed to stain in the dark for 10 min. This was followed by dilution to 1 ml. Samples were viewed under microscope a Nikon Eclipse 90i and a confocal D-Eclipse C1. Nile red produces strong orange fluorescence at 543 nm/598 nm (excitation/emission) and BODIPY produces a strong green fluorescence at 493 nm/503 nm.
Total lipids were extracted from samples of algal biomass essentially as described by Bligh and Dyer (1959). Lyophilised algal samples were weighed and placed in MP Biomedical 'lysing matrix E' (Cambridge, CB1 1BH, UK) tubes together with 1.5 ml sterile deionised water. Samples were beaten in a Fastprep FP120 beadbeater (Thermo Scientific Savant, Surrey, UK) at setting 6.5 at intervals of 45 seconds for a total of 3 minutes. Tube contents were removed via a pipette and rinsed with a CHCl3:MeOH (2:1) mixture. For each sample ~5 ml CHCl3:MeOH (2:1) was added and the sample vortexed. Samples were then centrifuged at 3000 rpm for 5 min. The bottom layer was removed to a round bottom flask. Two subsequent rinses were performed to remove the remaining oils by adding 1.5 ml CHCl3 to the sample, vortexing and centrifuging. Giving a total sample volume of 10 ml.
To transesterify the samples, MeOH (10 ml) and 18M H2SO4 (1 ml) were added to the sample, which was then held at reflux for 4 hours. The crude mixture was washed twice with H2O (50 ml) to remove the acid and resulting glycerol. The organic layer was isolated, the solvent was removed under vacuum and the samples were dried and dissolved in 2 ml of 1,4-dioxane (99 +%) and analysed by an Agilent 7890A Gas Chromatograph with capillary column (60m × 0.250mm internal diameter) coated with DB-23 ([50%-cyanpropyl]-methylpolysiloxane stationary phase (0.25m film thickness) and a He mobile phase (flow rate: 1.2ml/min) coupled with an Agilent 5975C inert MSD with Triple Axis Detector. The column was pre-heated to 150°C, the temperature held for 5 minutes and then heated to 250°C (rate of 4°C/min, then held for 2 min). The samples were quantified by comparison to known standards purchased from Sigma Aldrich.
The Roman Baths appear to have extensive microbial mat communities submerged beneath the water surface, some with striking blue-green patches. There are also filamentous 'hairy' brown mats that grow around evolved gas bubbles. These eventually create gelatinous balloons-like structures that rise to the surface, anchored to the microbial mat below by filamentous 'ropes'. A bright orange muddy residue, possibly caused by bacteria, detritus and iron hydroxide precipitates form a thick layer (~2-3 cm), between the mats and stone surfaces. In addition to iron, the spring water contains high concentrations of calcium, sulphate, sodium and chloride (Kellaway, 1991). There are also visible mineral deposits, which form dependant on water agitation, cooling and oxygenation (Kellaway, 1991). Water analyses of the Great Bath (GB) and Kings Bath (KB) show that the abiotic conditions in the baths have remained stable since records began in 1874 (see Additional file 1: Table S2, for full water analysis). The temperature of the Great Bath and Kings Bath were 39.0°C and 45.0°C respectively, measured 30cm below the surface.
In Figure 1, images of the isolates from the Roman Baths, alongside the reference alga Chlorella emersonii are shown. These are Chlorella emersonii (A), two green algae (B, C), a diatom (D) and four cyanobacteria (E, F, G, H). Isolates were identified as follows and will be referred to in figures in the same order by their abbreviations in brackets; B: Coelastrella saipanensis (Cs), C: Klebsormidium sp. (K sp.), D: Hantzschia sp. (H sp.), E: Chroococcidiopsis thermalis (Ct), F: Microcoleus chthonoplastes (Mc), G: Mastigocladus laminosus (Ml) and H: Oscillatoria sancta (Os). It should also be noted that there appears to be an abundance of microalgal species present in the baths, more than are discussed in this paper. Filamentous cyanobacterial species (O.sancta, M.chthonoplastes, M.laminosus) were isolated by from sample scrapings of microbial mats and contaminants removed using the 'agar plate scoring method'. All other species were isolated using the 'filter paper method'.
Light microscope images of algae isolated from the Roman Baths (x60). All algae were isolated from the Great Bath unless indicated (* = isolated from Kings Bath). A, Chlorella emersonii (Ce); B, Coelastrella saipanensis (Cs); C, Klebsormidium sp. (K sp.); D, Hantzschia sp. (H sp.); E, Chroococcidiopsis thermalis (Ct); F*, Microcoleus chthonoplastes (Mc); G*, Mastigocladus laminosus (Ml); H*, Oscillatoria sancta (Os). Bar = 20 μm.
Microalgae are often 'elastic' in their ability to alter their morphology based on their growth conditions. In a unique environment such as the baths, algae may potentially look different to species standards. In addition, it is not uncommon for algae which are distantly related to look similar and vice versa. As such identifying algae based on morphology is very difficult, therefore DNA barcoding was the preferred method for identification of isolated photosynthetic microorganisms. Isolates were assigned species or genus level identification using Basic Local Alignment Search Tool (BLAST) using the 'total score' values and in some cases images from online culture collections to confirm matches (see methods). In order to be consistent 'Total score' from the BLAST outputs was used as a means of identification. Any isolates with multiple hits of the same score, visual identification was used to help confirm an ID.
The isolate identified in Table 1 as C. saipanensis, initially received identical BLAST scores for Coelastrella saipanensis and Ettlia texensis. Upon closer examination both entries in the database comprised of identical nucleotide sequences. Despite the difficulty in identifying algae based on morphology alone, images from online culture collections were used in order to identify this isolate from the two identical database 'hits'. Images of Coelastrella sp. in online culture collections showed similar cell granularity ('speckled' inclusions) to C. saipanensis but did not show aggregates of diving cells inside mother cells (a feature of Ettelia spp.). Therefore has been identified as C.saipanensis. For K. sp. there were many similarly scored species of the genera Klebsormidium. However, there were no distinct physical features present to identify a species based on morphology.
Identifying H. sp. to the species level proved problematic. There were many very similarly scored matches from different groups of diatoms that are morphologically very distinct. Diatoms, unlike many unicellular algal groups, can be more easily identified using morphology alone to the expert eye. Identification of H. sp. was subsequently based on images from a slide preparation (described in methodology), which were subsequently assessed by Dr. S. Spaulding (Colorado State University) for identification. For M. chthonoplastes most of the outputs in the BLAST search regardless of sorting had a ~98% query coverage from uncultured clones and Microcoleus chthonoplastes strains (with max idents of 91%). However, Geitlerinema cf. acuminatum, had 77% query coverage (percentage of the input sequence covered by individual sequences in the database) but a 97% max ident score (maximum percentage of high scoring pairs in a segment). Little has been published on Geitlerinema spp. but a few culture collection images do more closely match the growth morphologies of the isolate (Mc) than Microcoleus i.e. M. chthonoplastes isolate grows as an amorphous tangle of filaments like Geitlerinema as opposed to discrete bundles of fibres like Microcoleus spp.
Roman Bath isolate sequences for Oscillatoria sancta and Microcoleus chthonoplastes were not accepted to the NCBI database due to alignment gaps and some chimeric sequences. This may be attributed to their very thick gelatinous outer cell walls which likely contain some contaminant bacteria. Both isolates require re-sequencing for more accurate species identification.
Results of the temperature tolerance experiments (Additional file 2: Table 2) show that the eukaryotic algae (C.emersonii, C.saipanensis, Klebsormidium sp. and Hantzschia sp.) behave similarly and are able to grow at 30°C but do prefer lower temperatures. The cyanobacterial isolates have more variation between them but all show good growth at 35°C and some growth at 40°C, unlike the eukaryotes. Based on these results further growth experiments were carried out at 20°C, 30°C and 40°C, to give a low, medium and high value for comparison.
During isolation and culture maintenance of the Roman isolates it was noted that O. sancta, C.thermalis and H. sp. showed sensitivity to the light intensity used to culture stocks of C.emersonii (200 μMol m-2 s-1) (data not shown). For this reason, it was decided to use a 'moderate' light intensity (80 μMol m-2 s-1) and shaking speed for temperature tolerance experiments. Although this allows for results to be comparable for a specific set of culture conditions, the culture parameters are likely to have been suboptimal for a number of the species examined (e.g. C.emersonii potentially extending to the other green algae K.sp, C.saipanensis and even the cyanobacterial isolates). Growth media may also be suboptimal.
To further examine the growth rate under nitrogen enriched conditions the microalgae were cultured over 12 days and sampled periodically. To clearly communicate this information the final dry weights are presented in Figure 2, all other data is presented in full in the Additional file 1: Figures S3-S5. These experiments supported the preliminary data, where the eukaryotic algae all accumulated biomass at the lower temperature of 20°C, were more productive at 30°C but generally did not grow at 40°C. K. sp. and C. saipanensis accumulated the most biomass at 30°C, comparable to the reference alga C. emersonii. Within the cyanobacteria, the single celled species C. thermalis showed better growth at lower temperatures. M. chthonoplastes, M. laminosus and O. sancta acquired the most biomass at 30°C, with M. chthonoplastes and M.laminosus showing better growth at 40°C than 20°C. This was not found to be the case for O.sancta whose growth was impaired at 40°C. M. laminosus acquired the most biomass of all the cyanobacteria, at a temperature of 30°C.
Growth of Ce and Roman Bath isolates in nitrogen sufficient medium after 12 days, measured as dry weight (g l -1 ).
Of all the algae tested, C. emersonii accumulated the most biomass at 20°C, K sp. the most at 30°C and M.laminosus the most biomass at 40°C. Both H. sp. and C. thermalis were not sufficiently productive at any of the temperatures investigated, achieving <0.2 g l-1 after 12 days, unlike other species which achieved between 0.5-0.9 g l-1 after 12 days.
Staining gave a visual indication of the neutral lipid products contained in a sample (Figure 3). Nile red stains lipids and fluoresces red, where the lipids are predominantly unsaturated and yellow where the lipids are saturated. Photosynthetic pigments including chlorophyll present in eukaryotic algae and cyanobacteria auto-fluoresce at the same excitation wavelengths as nile red. This can make it difficult to identify stained lipids against the autofluorescent 'backdrop' of the cell. However the fluorescence of these pigments is useful in assessing the health of the cell as stressed cells undergo breakdown of these pigments. This can be seen in Figure 3A and B showing nitrogen starvation of Klebsormidium sp. at 20°C. At 20°C K. sp. (Figure 3A and B) has clearly visible lipid droplets and a noticeable lack of chlorophyll. At 30°C however, during nitrogen starvation, K sp. shows little or no breakdown in chlorophyll or accumulation of lipids (Figure 3C and D).
Microscope images of Klebsormidium sp. cultivated at 20°C (A,B) and 30°C (C,D) and starved of nitrogen. Microscope images of Hantzschia sp. cultivated at 30°C with sufficient nitrogen (E,F) and starved of nitrogen (G,H). Stained with nile red. Bar = 20 μm.
Nitrogen starvation usually encourages accumulation of lipids in most microalgae (Cha et al. 2011). However, H. sp. at 30°C shows higher accumulation of lipids under nitrogen sufficient conditions (Figure 3E and F) compared to nitrogen starved conditions (Figure 3G and H). Staining images for all species are presented in the Additional file 1: Tables S3-S5, in full.
To assess the effectiveness of the microalgae to produce biodiesel, the neutral lipids from the microbial samples (the weight of which is given in Figure 2) were isolated, using a chloroform and methanol solvent extraction and then converted into FAME through acid-catalysed transesterification and analysed by GC-MS.
Reducing the nitrogen content in the culture medium for green algae generally increases the neutral lipid accumulation in the cell (Cha et al. 2011). This effect is illustrated by the reference species in Chlorella emersonii where the FAME produced from the sample increased from 23 wt% to 34 wt% of the dry weight under nitrogen starved conditions at 20°C (Figure 4). A similar effect was seen with K. sp. (increasing from 1.9wt% to 16wt%) and H. sp. (from 31.9 to 40.5%). In contrast nitrogen-depletion did not change the amount of FAME recovered dramatically for C. saipanensis. Irrespective of any other effect, the green algae produced less neutral lipid at higher temperatures. Nitrogen-starvation of cyanobacteria had a detrimental effect on the lipid percentage at both 20°C and 30°C. At 40°C, however, a high percentage of FAME (10.4-45.6%) was recovered from O. sancta under most conditions (with the exception of 30°C). This effect was not observed in M. laminosus, M. chthonoplastes or C. thermalis.
FAME produced from the Roman Bath isolates as a percentage of dry weight grown under A) nitrogen enriched and B) nitrogen starved conditions.
Reducing the nitrogen content in the growth media may have a detrimental effect on growth rate. Where this is the case, oleaginous microbes will fail to produce a substantial amount of biofuel on a per unit biomass basis (Figures 5 and 6). The highest biodiesel production was observed for C. emersonii and C. saipanensis at 20°C under nitrogen enriched conditions, where the lower content of glyceride lipids within the biomass were compensated by a higher growth rate. K. sp. and H. sp. biomass increased in the glyceride content when nitrogen content was reduced. None of the cyanobacteria produced a large amount of biodiesel irrespective of nitrogen conditions at 20°C and 30°C (Figure 5) compared to green algae C. emersonii and C. saipanensis (>0.15 g l-1 of biodiesel). For the most part, this was due to low levels of biomass accumulation in the cyanobacterial isolates (Figure 2). However, at 40°C, nitrogen reduction resulted in recovery of a larger quantity of biodiesel from the cyanobacteria, though this represented only a small amount of biodiesel overall.
Total amount of biodiesel recovered from the Roman Bath isolates grown under A) nitrogen enriched conditions and B) nitrogen starved conditions.
% change in the total amount of biodiesel produced on nitrogen starvation of the algal species.
The properties of biodiesel are highly reliant on the FAME profile (Fortman et al. 2008). Although chain length and the degree of unsaturation do have an effect on the fuel properties, the FAMEs can be placed into three loose categories to predict the fuel performance; saturated, monounsaturated and polyunsaturated esters (Figures 7 and 8). Biodiesel fuels rich in saturated esters have a superior cetane number but poor viscosity and low temperature properties; monounsaturated esters have acceptable low temperature properties and viscosity; fuels high in polyunsaturates have excellent low temperature properties and a low viscosity but a poorer cetane number and oxidative stability. Full FAME profiles were obtained for the isolated algae and are given in the Additional file 1: Tables S6-S8.
FAME profile of the microalgae cultured throughout this investigation.
FAME profile of the cyanobacteria MC and ML.
On reduction of the nitrogen content during the culturing of C. emersonii the FAME profile did not change dramatically. However, an increase in temperature, in both nitrogen regimes, resulted in an increased polyunsaturated component predominantly at the cost of the monounsaturated fraction (Figure 7). A similar outcome was observed for C. saipanensis. In contrast to C. emersonii and C. saipanensis nitrogen starvation of K. sp. increased the production of unsaturated FAME whereas both the nitrogen enriched 20°C and 30°C samples contained predominantly saturated FAMEs. Like the green algae C. saipanensis and C. emersonii, the FAME profile of H. sp. did not change significantly following nitrogen starvation. However, raising the growth temperature (20°C – 30°C - 40°C) increased the proportion of the saturated component. The relatively high level of saturates recovered form both K. sp. and H. sp. would result in a poor quality biodiesel.
The FAME profiles of the cyanobacteria were much simpler than those of the green algae. Unfortunately, insufficient biodiesel was recovered from O. sancta or C. thermalis to enable complete analysis of the FAME profile (see Additional file 1). M. chthonoplastes produced only saturated esters at both low temperatures and in nitrogen-rich conditions at 30°C (Figure 8). Nitrogen depletion, or an increase in temperature to 40°C, resulted in an increase in unsaturated esters and composition more suitable for biofuel. M. laminosus produced no polyunsaturated esters under any of the conditions examined. At low temperatures there were around 90% saturates which decreased to approximately 66% when the temperature was increased to 30°C, and to approximately 50% at 40°C. Nitrogen-starvation had little effect on the FAME profile at any temperature.
The excavation of the Roman Baths to uncover the present buildings and associated water-filled baths began in 1878 (Byrne 2008). Consequently, the opportunity for the colonisation of the baths by microalgae and other microbes has existed for some 134 years. Despite the city-centre location of the Baths and the relatively high water temperature initial sampling in the baths revealed the presence of multiple microalgal species. The isolates O. sancta, M. chthonoplastes, M. laminosus and H. sp., proved the predominant species and were repeatedly found when attempting to isolate other species. Whilst the present work isolated and identified 7 species, our efforts were not exhaustive and further species remain for future isolation.
The first step to identifying algae present in the baths was to establish axenic unialgal cultures. Whilst other algae are seldom difficult to remove, bacteria are often physically associated with the muciferous layer of the algal cell walls and are not always eliminated by standard serial dilution and plating techniques. Impurities in agar (derived from algae) can also prove inhibitory to the growth of some algae and may encourage growth of bacterial and fungal contaminants. Some cyanobacterial isolates grew poorly on agar most likely due to toxic component in the agar (Dworkin and Falkow, 2006). Micromanipulation, filtration and use of chemicals have been successful with some groups (Ferris and Hirsch, 1991). The use of antibiotics appears attractive but must be used in a narrow concentration range to avoid damage to the chloroplast in eukaryotes and lethality to cyanobacteria (Issa, 1999).
In order to amplify and sequence 16S (cyanobacterial) or 18S (eukaryotic) rDNA gene sequencing, the isolates needed to be subjected to some decontamination. Filamentous cyanobacterial species (O.sancta, M.chthonoplastes, M.laminosus) were isolated by from sample scrapings of microbial mats and contaminants removed using the 'agar plate scoring method'. All other species were isolated using the 'filter paper method'. However, it is evident that the microbial mat communities present in the Roman Baths are required for microbial growth and survival. Hence it is also highly likely that resident algae have formed relationships with other organisms present (Croft et al. 2006). Although most of the contaminants have been removed, the isolates are described as unialgal.
Employing a DNA-barcoding approach utilising the U16S and U18S rDNA gene sequences and a NCBI BLAST search as an identification tool proved problematical for M.chthonoplastes, C.saipanensis, Hantzschia sp. and Klebsormidium sp. This appears to arise due to both a relative paucity of gene sequences from microalgal species and the presence of incorrectly assigned sequences due to poor regulation of the NCBI database. Naturally, there is also a substantial bias toward more commonly used species. There is still uncertainty about the optimum fragments of DNA to use for identification (Surek, 2008). Cox1 is the standard for most 'higher animals' but is not suitable for green algae and higher plants because its rate of evolution is too slow (Surek, 2008). Common gene targets for algal identification are rRNA genes, mitochondrial genes and plastid genes, but each may fail to provide a conclusive result. Clearly there is a need to identify a single universal short DNA fragment that gives a clear identification of species (Surek, 2008).
Differences in geographical location can result in changes in target sequences within a species. This has been demonstrated for M. chthonoplastes (Garcia-Pichel et al. 1996) and Synechococcus sp. (Miller and Castenholz, 2000). Whilst this type of analysis has provided evidence that more thermotolerant lineages evolved from less thermotolerant ancestors (Miller and Castenholz, 2000), such ecologically induced changes can further complicate DNA sequence-based species identification.
Where DNA-barcoding proved inconclusive, algae were subjected to morphological examination at the light microscope level and images compared to online culture collections (e.g. the Culture Collection of Autotrophic Organisms (CCALA)). This approach proved successful for C.saipanensis but not for H.sp. Microalgae often lack distinct morphological features that can make them hard to identify (Pulz and Gross, 2004). Moreover, algal development can be plastic with cells changing shape and size during the lifecycle and in response to changes in culture conditions (Cheng et al. 2011). Consequently, algal identification and taxonomy demands a 'polyphasic' approach that uses both molecular and morphological data as employed in this study (Surek, 2008).
Cyanobacteria are often found in warm, low nutrient environments (De Winder et al. 1990), and are typically more thermotolerant than eukaryotic algae (Barsanti and Gualtieri, 2006). Members of the single-celled cyanobacterial genus Thermosynechococcus are capable of surviving at 73-74°C, whilst thermophillic filamentous cyanobacteria typically occupy a lower temperature range of 55-62°C (Seckbach, 2007). In accord with this the present study found 50% of the isolates from the Roman Baths were cyanobacteria, and that these species remained viable and productive at higher temperatures than the eukayotic isolates.
Eukaryotic algae are generally absent from environments above 56-60°C largely due to the instability of organellar membranes at high temperatures (Tansey and Brock, 1972). Members of the Rhodophyta such as C. caldarium have been found to withstand 57°C (Seckbach, 2007). However, none of the Roman Bath isolates were from this genus. The comparatively poor growth of the eukaryotic isolates compared to the cyanobacteria, suggests that they were present in cooler locations within the baths i.e. on exposed surfaces close to the waterline.
In some cases there was a lack of correlation between growth rates at high temperatures in vitro and the prevalence of a particular species in the Baths. For example, O. sancta showed limited growth at 40°C (Figure 2) despite being the dominant microalgal species in the baths where it forms filamentous 'balloons' around evolved gas bubbles. In both environments the species is brown in colour rather than the typical blue-green. This is consistent with previous studies showing that high light or high temperature result in Oscillatoria sp. making a similar colour change and an associated reduction in growth rate (Gribovskaya et al. 2007). To ensure comparability of temperature effects all other conditions were moderated in order to be consistent for all species tested. For example, C. emersonii exhibits a faster growth rate at a higher light intensity over longer light: dark cycles, however during culturing these conditions were found to be detrimental to growth rate for C. thermalis, H. sp. and O. sancta (data not shown). Growth was therefore highly likely to be suboptimal for all the species investigated and further work is required to determine optimal growth conditions.
The poor performance of unialgal cultures may reflect a requirement for cooperation in a microbial community. Microbial mats are important primary producers in extreme habitats where they provide environments that facilitate survival (Pattanaik et al. 2008). Evidence also suggests that cultured communities are more productive than monocultures (Croft et al. 2005). Microbial mats are prevalent in the Roman Baths and may therefore enable species to survive in this high temperature environment. The Roman Baths contain good levels of most trace elements required to support growth. However, some of these nutrients are in large excess which in turn can cause stress to algal cells. Calcium, sodium and chloride levels are much higher than standard media used for culturing these groups of algae (Barsanti and Gualtieri, 2006) and iron levels are much higher in the baths than most freshwater sources (Kellaway, 1991). In many aquatic environments iron is a limiting nutrient for productivity, yet in the baths it is at a similar concentration to most growth media. However it is important to note that for some species this could represent an excess. High iron concentrations have a negative effect on phytoplankton growth and increase oxidative stress (Estevez et al. 2001). Silicon is also plentiful in the bath, which presumably accounts for the abundance of H. sp.
Although nitrogen starvation and other stresses trigger neutral lipid accumulation in eukaryotic microalgae this is generally associated with a reduction in growth rate (Illman et al. 2000). This reduction was observed for C. saipanensis (Figure 2), where more FAME was produced under nitrogen starvation on a per cell basis (from 14.3 to 14.9 wt%) but had a low total productivity of biodiesel due to limited biomass production under these conditions (Figure 5). This trade off is therefore an important consideration when selecting microalgae for a particular production regime. Interestingly, although the cyanobacteria examined in this work accumulated a lower diversity of FAMEs than the eukaryotic algae, these species produced high levels of neutral lipids that were converted into FAME (up to 45 wt%) under nitrogen-rich conditions (Figure 5). The fact that cyanobacteria can accumulate lipids in the thylakoid membrane under conditions promoting photosynthesis and high growth rates may explain this behaviour (Karatay and Dönmez 2011).
The green algae C. saipanensis produced a large amount of polyunsaturated esters under all the conditions trialled similar only to the reference algae C. emersonii (refer to data). This FAME profile is roughly equivalent to sunflower or soybean oil (Knothe et al. 2005) and the resulting biodiesel would therefore have similar fuel properties including relatively high oxidative instability. Biodiesel produced from H. sp. and K. sp. was more saturated than that obtained from the other microalgal species (Figure 7). This effect increased with rising temperature (for H. sp. and K. sp. and increase in temperature from 20-30°C increased saturates by 14.0% and 13.2% respectively). Fuels high in saturates tend to have high cloud points, poor low temperature behaviour and a high viscosity (Knothe et al. 2005). Consequently, biodiesel produced from H. sp. and K. sp. would be unsuitable for high blend levels but would have combustion qualities akin to high performance diesel fuel such as a high cetane number, lower NOx emissions and be highly oxidatively stable.
The cyanobacteria M. chthonoplastes and M. laminosus were rich in saturated esters (Refer to data). Although difficult to generalise, cyanobacteria tend to produce saturates in larger quantities than other species especially when cultivated at higher temperatures (Balogi et al. 2005; Nanjo et al. 2010; Dinamarca et al. 2011). Since unsaturated esters are more oxidatively stable this may be a direct response to a high temperature environment. Interestingly, the levels of saturated esters isolated from M. laminosus were reduced when cultured at higher temperature and at 40°C the biodiesel produced was almost 50% monounsaturated. Monounsaturated esters on balance have the most promising fuel properties, and biodiesel rich in these esters, such as rapeseed methyl ester or olive oil methyl ester, can be used at higher blend levels than other types of biodiesel so with careful control of the culture conditions biodiesel with suitable physical properties can be produced from the algal species isolated.
Our aim was to identify thermotolerant, oleaginous microalgae with potential as a source of renewable biodiesel. The Roman Baths proved to support a rich diversity of microalgal and cyanobacterial species. A total of 3 green algae, 1 diatom and 4 cyanobacteria were successfully established as unialgal cultures, and the majority assigned a species identification using a DNA barcoding approach. Whilst, a number of species produced high levels of neutral lipids, suitable for FAME production, under a range of conditions, all were more productive at lower temperatures than found in the Baths. Whilst these species do not sustain high productivity at extreme temperatures, an ability to survive temperature spikes in an open pond production system is an extremely desirable trait (Pulz and Gross, 2004). To date, the few species that are successfully cultivated commercially in open ponds are extremophiles able to grow in a highly selective environment (Xu et al. 2009). This work highlights the diversity in form, products and behaviour of algal species isolated from the same extreme environment and the importance of screening for new species. However a rapid species screening method requires a quick and efficient extraction method, which does not affect FAME profiles and ideally is scalable.
The culture conditions used to screen the microalgae were not optimised, suggesting that some of species could be developed into effective biodiesel producers. Four species, K. sp., M. chthonoplastes, M. laminosus and O. sancta, are filamentous, which could reduce harvesting and dewatering costs. One of these, M. laminosus, is also nitrogen-fixing species, which could again reduce input costs. Both these features could assist process on scale-up.
We would like to thank both Johnson Matthey and the DENSO Corporation for help both financially and materially, and Roger Whorrod for his generous endowment to the University resulting in the Whorrod Fellowship in Sustainable Chemical Technologies held by Dr. C. Chuck. We would like to thank Tom Byrnes and all the staff at the Roman Baths, Dr Miles Davis, Severn Trent Services for help with the water analysis and Dr. Sarah A. Spaulding of Colorado State University for help in identifying the diatom H. sp.


Left Back Manc is an Au Pair who has been living in Paris for three years. She writes the anonymous but successful blog Left Bank Manc (www.leftbankmanc.blogspot.co.uk) and very kindly agreed to let us know a little bit about her adventures as an Au Pair.
The fact that I didn't speak French and had never visited the city before wasn't a factor in my decision making, although I was realistic enough to realise that it wouldn't be easy finding a job and somewhere to live. I decided to try Au Pairing because it seemed like the best option. I loved children, families were looking for native English-speakers and I wouldn't have to worry about my accommodation.
Being an Au Pair is a great way to live in a new city and experience another culture. Although my first Au Pair family didn't work out (they didn't want me to be out after 10pm and the few 'fun activities' I was allowed to do with the kids included walking for miles on end... and an abacus), I have since had fantastic experiences with two other families.
Lodging with a family is a nice idea for girls fresh out of sixth form college but for people like me, who had already moved away from home to go to university, it's a much better option to get an Au Pair job with separate accommodation.
After I moved on from that first family and found a job that came with an independent studio, my year in Paris was just how I imagined it would be. I started to make friends with other nannies through the 'Au Pairs in Paris Facebook' group, and also through my blog, and we would all take our kids to the park after school. I explored the city and enjoyed my job. It wasn't always easy. The three kids could be bratty and stubborn. But at the end of a difficult day I could go to a bar with my friends, drink cheap wine and forget all about the five year old refusing to brush his teeth.
Despite the spoilt kids, neurotic parents and living in a room where I could theoretically shower and cook spaghetti at the same time, I would definitely recommend Au Pairing. The experience allowed me to live in Paris rent-free, with a job that is both rewarding and fun (most of the time).
My current Au Pair family only need me for two hours each evening, meaning that last year I was able to take on a second job in a restaurant (which vastly improved my French) and this year I've found plenty of well-paid teaching work to keep me busy. Not many people choose to work as an au pair for as long as I have, but it's a great idea for a short career break and you never know, that year out might turn into three.
I started a blog called Left Bank Manc so I could share my awkward stories and experiences of Parisian nightlife, which led to me becoming a kind of Au Pair Agony Aunt, with girls often asking me for advice. This can range from how to tell the family they can't eat any more courgette soup, to what to do about the sinister neighbour who broke into their room and stole a pair of knickers etc.


Taking a break from Dark Days Challenge meals this week because I'm still recovering from a fantastic Valentine's Day meal and there are tons of leftovers in the fridge. The kitchen partner's baked trout rocked my world last night. We don't have many options in Minnesota to feed my seafood cravings, but he always finds a way to make a special meal work. As many times as I salivate over that pile of once-frozen shrimp in the meat case, I have serious qualms about eating seafood. A local baked trout is as good as it gets.
My concerns about seafood began in 2010 after watching The End of the Line, a documentary examining our global struggles with over-fishing. While I question some of the calculations (can we really measure if current rates of overfishing will result in the end of seafood by 2048?), it was hard to argue with the images. I was a sushi lover before I saw this film and now I rarely indulge. Tilapia and salmon were my go-to restaurant choices, thinking I was making a better "eco" decision than eating beef. Now I better understand that our food system is decimating global fish populations; regardless of how many years it will take, it is clear our cravings for seafood are unsustainable.
The End of the Line left me confused about eating fish in general. The choices I assumed were better, like fish over eating beef, eating farm-raised over wild-caught, are in fact not always the most sustainable. Local beef may have fewer harmful environmental impacts than a wild-caught tuna from the Southeast Asia. Likewise, farm-raised fish have to eat something, and in many cases it's smaller, wild-caught species. Take tilapia as an example. Supposedly, we farm-raise tilapia because wild-caught supplies cannot meet the demands of the population. But farm-raised tilapia and other larger species are fed fish meal, made from smaller species. Does it really make sense to over-fish sardines to feed farm-raised tilapia, grown only because it too is over-fished?
Since the documentary I have relied heavily on The Monterey Bay Aquarium's Seafood Watch program to make better informed decisions about seafood. Monterey's consumer-education program details which species are best to consume and which should be avoided. I've used both their downloadable wallet-sized guides and the handy app available for iPhone and Android. Each species are categorized by best, good, or avoid and divided out by region. Minnesota is in the Central U.S. guide which includes many of the fresh water Great Lakes fish. Ever wonder what to order when you're vacationing on the North Shore? This guide is your best support in making a sustainable choice.
Which brings us back to baked trout. The guide lists Lake Michigan lake trout in the "avoid" column and Lake Superior and Huron trout as "good" choices. The "best" choice for trout is U.S. farmed and locally sourced. Exactly what we had for Valentine's dinner last night. There are several trout farms within driving distance of the Metro; check out: Valley Springs, Star Prairie, and Tallakson's.
1. Pre-heat oven to 350° F. Prepare a baking dish with non-stick spray (choose a dish that is large enough for the fish to lay flat in). In a small bowl, combine sugar and spices. Gently rub spice mix on fish, ensuring an even coat on both sides. Place fish in the baking dish. Bake 20-25 minutes or until fish flakes easily with a fork.


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CITES: Keep Calm And Let Africa Speak
November 2016 - Volume 14-4 & 5
September 6, 2018 In November 2016 - Volume 14-4 & 5 By CIC CITES Team & Gerhard R Damm
By CIC CITES Team & Gerhard R Damm
The International Council for Game and Wildlife Conservation, CIC, sponsored two high-level panel discussions and press conferences on 24th and 28th September during the 17th Conference of the Parties of CITES. Initially CIC planned only one press conference, but due to demand by the Africa Ministers present in the first meeting and the interest generated, a second event was scheduled.
Three African environment portfolio ministers, The Honorable Edith Edna Molewa, Minister of Water and Environmental Affairs (South Africa), The Honorable Pohamba Shifeta, Minister of Environment and Tourism (Namibia), and The Honorable Oppah Chamu Zvipane Muchinguri, Stephen Mwansa, Permanent Secretary Ministry of Tourism & Arts (Zambia), Prince Mupazviriho Chiwewete, Permanent Secretary Ministry of Environment Water and Climate (Zimbabwe), Shonisani Munzhedzi, Deputy Director-General: Biodiversity and Conservation of the Department of Environmental Affairs (South Africa), Willem Wijnstekers, former Secretary General of CITES and Deputy President Division Policy & Law of the CIC International Council for Game and Wildlife Conservation (The Netherlands) and Wilfried Pabst, owner of Sango Conservancy & Lodge within Savé Valley Conservancy (Zimbabwe) formed an impressive panel. With ingrained authority the panelists addressed the topic "Keep Calm and Let Africa Speak" and took questions and comments from around 100 guests and journalists from around the world in the two meetings.
The discussions and interactions with the plenum was moderated with distinction by Dr. Ali A. Kaka, Africa Regional Councilor for the International Union for Conservation of Nature (IUCN).
The participating journalists packed the press room to hear firsthand the views of the African representatives on the proposals being submitted to and discussed at COP 17. It soon became clear that the panelists considered many of the proposals to be primarily driven either by African States with very poor conservation results or by non-African states which have little or no wildlife of relevance to the proposals up for decision by the voting delegates at CITES.
Clearly aggrieved at many suggestions on which they were not consulted as range nations, and/or on which their views based on successful conservation models were not taken with the seriousness they deserved the panelists did not mince words and spoke candidly.
The audience was accorded ample time to ask questions and make observations. Keen observers noted that there was widespread support for the sentiments of the panelists. Questions at some of the panelists on the status of their wildlife and on some incidents that intended to cast a shadow on the national wildlife management strategies of the participating countries were answered with resounding arguments.
In his opening remarks Dr. Ali Kaka got right to the point saying "We are talking about the sustainable use of wildlife".
Namibia's Minister Pohamba Shifeta, for example, pointed out that friendly coexistence is a myth and that there is a price communities living in close proximity with dangerous wildlife pay. Namibia boasts of community conservancies established under enlightened legislation that allows people to benefit economically from the sustainable use and conservation of wild animals. Conservation hunting in Namibia is based on sustainability, and the proceeds go to the community conservancies. Therefore the communities understand the value of wildlife. Shifeta appealed to the listeners (and implicitly also to the greater community of CITES delegates) when he concluded: "Don't be influenced by emotions! First and foremost you need to comprehend the point of view of our rural communities, who live from and with wildlife!"
Namibia's Minister also mentioned that those living on other continents should stop prescribing Africans what to do with African wildlife. "They are infringing on the sovereignty of African states", Shifeta said. Participants could sense similar indignation from the representatives of South Africa, Zimbabwe and Zambia, as well as from private conservancy owner Wilfried Pabst. Stephen Mwansa of Zambia stressed that "wild animals are there because of our conservation efforts and that includes their consumptive use!
"We're being told by people outside, by people my color of skin, how to run things in Africa," Wilfried Pabst, owner of the Sango Conservancy in Zimbabwe, said. He highlighted that the call for a ban earlier this year by some European Union parliamentarians on the importation of trophies was equivalent to banning hunting. "They have neither regard nor understanding of what they are effectively destroying. In southern Africa alone, if we ban sustainable use, we would eliminate some 55 million hectares of land under conservation and lose in the region of 20 million animals, hundreds of thousands of jobs, each man or woman supporting a family of 10, thus putting millions of local people into destitution," Pabst passionately explained. "Sustainable use is a "very successful conservation model", he said.
"Listen to me very carefully, I mean every word I'm saying," Pabst continued emphatically, "untold NGO's do good work, but there are those who are against the sustainable use of wildlife resources, those who are disguising the intention of banning sustainable hunting behind the mask of wanting to forbid the importation of trophies into Europe and the USA."
"I declare with conviction that the NGO's directly or indirectly propagating to end sustainable hunting, that these NGO's are criminal organizations. Did you hear this? Criminal. Because they are soliciting money under false pretenses – this is fraud and thus criminal. None of the donors are told, as they should be, that sustainable hunting is one of the two largest contributors in funding conservation globally. Taking the income from sustainable hunting away will destroy some 75% of all wildlife areas in southern Africa alone. If the well meaning donors would understand that these NGO's business plans they are funding would cause the destruction, they would not donate. They are kept in the dark that their funds are actually funding the exact opposite of what they believe their contribution would achieve."
"None of these NGO's would like to see an intact and growing Wildlife scene. Why? If Wildlife is well conserved as in southern Africa at large, these NGO's could not raise any funds, their business model would die, their offices, vehicles overseas trips would disappear. Conclusion? These
NGO's can only survive or thrive by leaving donors in the dark as to the real effect of their donations and they need chaos and destitution in wildlife conservation to solicit these funds. That is indeed a criminal business model," Pabst concluded.
Prince Mupazviriho Chiwewete supported Pabst, saying Zimbabwe had not received a single cent of the millions of dollars collected for Cecil the Lion. "When decisions are made without consulting us, without taking into consideration our interest, those decisions are not made in the interest of conservation" he concluded. Shonisani Munzhedzi (South Africa) added that "…local communities are the ones who have to be involved in decision making that affects them! They should be assisted and supported, and we need to ascertain that they benefit!"
"Sustainable use, conservation and fair equitable benefit sharing are the pillars of the natural resources! There is no way that we would have sustainable conservation without looking at the issues affecting people, " Munzhedzi concluded.
The Honorable Minister Oppah Chamu Zvipane Muchinguri (Zimbabwe) reinforced these statements by saying "it is easy for those not living with the elephants and other dangerous wildlife to preach and act noble. Yet it is our people who have to bear the cost of this existence. Removing incentives for these communities is to condemn them to perpetual poverty none can understand unless you have experienced it…." She also noted that "it was sad some governments and NGOs are failing to appreciate that sustainable use of wildlife is a key pillar to successful conservation. Communities as major custodians of wildlife resources need to continue accruing economic benefits from wildlife, since otherwise they would have little incentive to conserve it."
The Honorable Edith Edna Molewa, Minister of Water and Environmental Affairs (South Africa) mentioned already in her opening address to the CITES delegates that "our commitment to the sustainable utilization of natural resources contributes significantly to socio-economic development of poor and rural communities as part of our country's economic and social development."
Steven Mwansa passionately summarized the position of the panelists, "Please leave us alone!"
The CIC International Council for Game and Wildlife Conservation is a politically independent and globally active advocacy working in the public interest with a diverse membership that includes states, corporates, universities and associations. The core strength of the CIC rests in dedicated hunter-conservationist members from all continents. CIC advocates wildlife conservation across diverse landscapes through shared incentive-driven use. Our adaptive management approach is grounded in cutting-edge science. CIC values traditional knowledge reflected in millennia of diverse hunting heritage. The members uphold and evolve the fundamentals of ethical hunting. CIC aims that each generation bequeaths to its successor intact landscapes richer in wildlife.
The CIC CITES Team consisted of Heli Siitari, President of CIC Division Policy and Law, Willem Wijnstekers, former CITES Secretary General & Deputy President of CIC Division Policy and Law, Willy Pabst, CIC member and conservancy owner in Zimbabwe, Tamás Marghescu, CIC Director General.

redpajama_set_name: RedPajamaCommonCrawl


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Q: inverse Discrete-Time-Fourier-Transform of $F(\Omega) = \frac{1}{1+a^2-2a\cos(\Omega)}$ Find Inverse Discrete-Time Fourier Transform of:
$$F(\Omega) = \frac{1}{1+a^2-2a\cos(\Omega)}~~~~~~~~|\Omega| < \pi$$
What's the technique for finding the DTFT of the above function?
If I want to do this the quick way, is there a DTFT table entry for this type of Function?

A: Lets simplify the expression:
$\cos(\Omega)=\frac{e^{j\Omega}+e^{-j\Omega}}{2} \rightarrow F(\Omega)=\frac{1}{1+a^2-2a\cos(\Omega)}=\frac{1}{1+a^2-ae^{j\Omega}-ae^{-j\Omega}}=\frac{1}{(a-e^{j\Omega})(a-e^{-j\Omega})}$
Now we can use the the convolution properties of Fourier.
$x[n]*y[n]=X(\Omega)Y(\Omega)$
As a result:
$x[n]=\frac{1}{a}a^n u[-n]$ and $y[n]=\frac{1}{a}(\frac{1}{a})^nu[n]$ according to the Fourier table. (Verify my calculation)

redpajama_set_name: RedPajamaStackExchange


With a heart – stopping voice & wide – ranging instrumental talent, Odetta Hartman carries cowboy soul into an era where country can clash with computers, and bluegrass isn't afraid of bass. Her debut LP 222 — an experimental, bedroom-produced hybrid of folk, musique concrète and psychedelia, was released on Northern Spy Records in 2015. Alongside her partner, sound artist and producer Jack Inslee, Odetta continues to explore the sonic vernacular created by their uncanny collaboration: badass banjos, detuned violins, field recordings, superstitious soundscapes, and vocal stylings ranging from sensual to spooky.


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Michael Avenatti: 'Complete garbage' to blame me for Kavanaugh
'See Steve Bannon's comments on Bill Maher a week ago. That tells you everything you need to know'
October 8, 2018 | 12:01 pm
LAS VEGAS, NV – AUGUST 31: Attorney Michael Avenatti arrives at a news conference with Battle Born Progress, a progressive communications organization, on August 31, 2018 in Las Vegas, Nevada. Avenatti is representing adult film actress/director Stormy Daniels in her cases against U.S. President Donald Trump and his former attorney Michael Cohen. (Photo by Ethan Miller/Getty Images)
By	Cockburn
https://spectator.us/topic/michael-avenatti-kavanaugh/
Michael Avenatti says 'threatened' politicians who blame him for the Senate's decision to confirm Brett Kavanaugh to the Supreme Court are talking 'complete garbage'.
The lawyer, who rose to national prominence by representing the adult film actress Stormy Daniels in her suit against the President, produced the testimony of Julie Swetnick, the third woman to accuse Justice Kavanaugh of sexual misconduct. She claimed that Kavanaugh and his friend Mark Judge were part of a group who regularly spiked the punch at parties so that women could be gang-raped. Her allegations came under scrutiny following her interview with NBC News.
Sen. Gary Peters, a Michigan Democrat, said, 'I think we should have focused on the serious allegations that certainly appeared very credible to me that would be our best course of action.'
And a Democratic senator on the Senate Judiciary Committee complained to CNN: 'It wasn't helpful because the story became about Avenatti.'
But Avenatti said, 'not a single vote was impacted by my involvement.
'Collins stated she did not believe Dr Ford – end of story. This myth is being advanced by politicians that are threatened by me and rightfully so.'
Is that really the 'end' of the 'story' though? The no-nonsense lawyer appears to be overlooking the following part of Sen. Susan Collins's address to the Senate:
'Some of the allegations levied against Judge Kavanaugh illustrate why the presumption of innocence is so important. I am thinking, in particular, not of the allegations raised by Professor Ford, but of the allegation that when he was a teenager, Judge Kavanaugh drugged multiple girls and used their weakened state to facilitate gang rape. This outlandish allegation was put forth without any credible supporting evidence and simply parroted public statements of others. That such an allegation can find its way into the Supreme Court confirmation process is a stark reminder about why the presumption of innocence is so ingrained in our American consciousness.'
Avenatti clarified to Cockburn that he thinks both Republicans and Democrats are threatened by him.
'See Steve Bannon's comments on Bill Maher a week ago. That tells you everything you need to know,' he said, referring to the former White House chief strategist's thought that the lawyer had a 'fearlessness' which means he could 'go through a lot of that field, if he decides to stick with it, like a scythe through grass.'
A Democratic Senate aide told Vanity Fair: 'Every time Michael Avenatti's name comes out, it hurts. He is toxic and counterproductive and should go away.' If, as is rumoured, the lawyer is considering a 2020 run, he may have some bridges to mend with these 'threatened' Democrats to earn their backing…
Mischief, mayhem and Washington gossip. Send tips to cockburn@spectator.us


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Q: jquery выбор элемента в котором значение атрибута содержит спец. символ (точку) Всем привет!
Есть Asp.net SPA приложение написанное на C#, в нем используется клиентская часть написанная на MarionetteJS.
Внутри View Marionette я использую функцию которая извлекает div c помощью jquery.
html код поля ввода :

Если я в коде ищу элемент div  так:
var element = $(".session-status div[data-session-id=1234\\.1b50\\.5887]");

то div :

нормально извлекается .
Но мне нужно в атрибуте data-session-id убрать точки и лишь потом сделать выбор div т.к
jquery такой селектор
$(".session-status div[data-session-id=1234.1b40.5887]");

не понимает и выдает ошибку из-за наличия точек их я так понял нужно экранировать слэшами.
И вот когда я привожу значение атрибута в нормальный вид выбор элемента не происходит.
Вот мой код :
    var id = "1234\\\\.1b40\\\\.5887";
    var selector = ".session-status div[data-session-id=" + id + "]";
    var firstsdiv=$(selector); //НЕ работает
    var secdiv =$(".session-status div[data-session-id=1234\\.1b40\\.5887]"); //Работает

ошибку выдает такую:
jquery.js:1458 Uncaught Error: Syntax error, unrecognized expression: .session-status div[data-session-id=1234\\.1b40\\.5887]
    at Function.Sizzle.error (jquery.js:1458)
    at Sizzle.tokenize (jquery.js:2075)
    at Sizzle.select (jquery.js:2479)
    at Function.Sizzle [as find] (jquery.js:855)
    at jQuery.fn.init.find (jquery.js:2695)
    at new jQuery.fn.init (jquery.js:2803)
    at jQuery (jquery.js:76)
    at child.onGetSession (request-network-services.js:314)
    at HTMLDivElement.dispatch (jquery.js:4435)
    at HTMLDivElement.elemData.handle (jquery.js:4121)

Подскажите пожалуйста в чем ошибка , вроде бы селекторы выбора одинаковые получаются , видимо jquery по своему работает с экранируемыми символами.
Видимо тут подскажут только специалисты по C# которые еще и знают JS. Потому что на jsfiddle.net код
var id ="1234\\.1b40\\.5887";
var elm = $(".session-status div[data-session-id="+id+"]");

Работает . Скорее всего проблема с тем что приложение размещено на IIS и он как то преобразует строки по своему

A: Поставьте кавычки:

console.log($(".session-status div[data-session-id='1234.1b40.5887']").length);

    TEST 1

TEST 2

A: Рабочий селектор такой (просто добавил кавычки):
var id = "1234.1b40.5887";
var selector = ".session-status div[data-session-id='" + id + "']";


text:
Mysteries of the Unexplained: Bacon
Jun 09, 2009 by Andy Horwitz	in Theater
Mysteries of the Unexplained: Bacon by Mike Daisey, Joe's Pub, June 8, 2009
Review by Rachel Kramer Bussel
When monologist Mike Daisey took the stage at Joe's Pub last night for Mysteries of the Unexplained: Bacon, there was a collective, though silent, sigh. There was no bacon in sight. Since every other show I've seen him in has just been him at a table, with some paper and a glass of water, I'd assumed we'd see the same thing, but with him frying up some bacon. He let us marinate in our collective hunger for bacon before letting us know that instead of doing double duty, he'd gotten Obie Award-winning playwright Heidi Schreck to fry the ten pounds of bacon, adorned in a cute apron. The show's promo promised that "we'll explore bacon in all its filthy, gorgeous deliciousness," and I think it's fair to say Daisey succeeded. More than in other shows I've seen him do, this time he spoke directly to the audience, for a "communal celebration" as we jointly worshipped at the "church of pork," implicating us in our love of bacon. He spoke of hipsters in Williamsburg, and how as much as we want to crucify them (while they're wearing "I'm Being Crucified" ironic t-shirts), we also love them, perhaps because we all share a love of bacon.
Heidi Schreck practices her bacon-frying skills; photo via Mike Daisey's blog
He explained that while he had originally thought he could be the one frying the bacon and deliver his monologue, that just wasn't possible. He picked Schreck, who he'd worked with in Seattle, seemingly because she didn't have much culinary experience. As for the bacon, it wasn't gourmet. "Ladies and gentlemen, in the style of myself, we just have a lot of bacon," said Daisey. Upon announcing the theme of the show, Daisey was bombarded with emails about bacon. "You have to talk about this in the show," people said, when really, in Daisey's words, they were just showing him things they wanted to eat, or at least, discuss. Like the Bacon AK 47.
via This Is Freaking Ridiculous
Along the way to telling the story of how he came to be on a South Pacific island [Tanna – read more about it here] and faced with a wild pig he's supposed to kill, Daisey mused on his sexuality. "It was clear I could have a real life in the bear community–this was a fetish I could pull off." Alas, "I was just straight and loved bacon." Bacon became something that Daisey, and his audience, collectively salivated and obsessed over, listening to the monologue while sneaking glances at Schreck, who mostly kept to her cooking duties, occasionally responding to a slight verbal jab from Daisey. Daisey noted that he wished he were part of one of the religions that bans eating bacon, if only to defy those proscriptions.
He extolled the wonder of the bacon at Williamsburg steakhouse Peter Luger, calling it the best bacon. He also touched on a subject near and dear to my heart and my stomach: cupcakes, linking them to bacon as a food that's been hipsterized and fetishized, suggesting there might be a shop opening up to sell only artisinal bacon called Sizzle, and that there'd be meetup groups to jointly cook it (yes, my cupcake blog has a meetup group, thank you very much).
But the power of Daisey's shows, this one especially, isn't about pointing to or laughing at his audiences, but about joining them to him. By choosing such a communal topic as bacon (suggested by an audience member at the first Mysteries of the Unexplained, which was about Facebook), he was able to make us think about why, exactly, we love bacon so much, while also letting us know that, as fatty and forbidden as it may be by religions and health nuts, it's not just okay to like it, it's almost imperative. As for me, I am not a bacon fanatic, but I do enjoy it, and have found that, like cupcakes, it is a word that incites an immediate reaction. Daisey played this to good effect, and while the topic wasn't as personal or intense as some of his previous work, it was also more fun. How could it not be when the sparseness of his set was brightened by a woman meticulously cooking bacon for an hour?
At one point in the show, my friend Heidi whispered to me, "It's sizzling." And indeed it was. The smell of bacon began permeating Joe's Pub and distracted just a bit from Daisey's words. I had eaten a dish of meatballs, along with some of the ham Heidi gifted me from her salad; she is one of those vegetarians who love bacon so much they just can't stay away (I once gave her a bacon wallet as a gift). Thus stuffed (I also had sautéed spinach, and it was all delicious), I wasn't too too disappointed when at the end of the night, after settling our bill, we found that the bacon that had been fried up by Schreck and handed out by Daisey's wife/director Jean-Michele Gregory, was gone. We were treated to a (wrapped) package of bacon-flavored jelly beans, and Bacon vs. Tofu action figures. "We're going to smell like bacon," Heidi warned me as we exited. I'm not sure if we did, in fact, smell like bacon for the rest of the night, but I do know that if I did, I, like Daisey, don't mind a bit.
The next Mysteries of the Unexplained will be July 6th at Joe's Pub. See Mike Daisey's website for forthcoming show topic, to be picked from audience suggestions.
Tagged with: bacon, Joe's Pub, Mike Daisey, rachel kramer bussel


Home » Useful Guide » How to Convert Sony HDR-SR10E MTS to FLV for Web Sharing?
Do you have some short films shot with Sony HDR-SR10E AVCHD camcorder, and want to share them on your website? It will make your site more attractive with videos. While Apple competes with Adobe, the Flash support on Apple devices like iMac, MacBook Pro do not support exporting to Flash FLV/SWF now. You can not directly export FLV from the Apple QuickTime Player, iMovie or Final Cut Pro/Final Cut Express.
One solution is to upload videos to YouTube and embed YouTube videos on your site. If you do not want to embed YouTube videos, you can convert Sony HDR-SR10E MTS files to FLV with Mac Sony MTS converter, and put the FLV videos on web server with Flash player. Aunsoft MTS/M2TS Converter for Mac is the recommended Mac Sony HDR-SR10E MTS converter for you. It exports Flash videos with automatic deinterlacing function, which makes the FLV videos play without interlacing artifacts. It could also help you merge several Sony .mts video clips and export as one file.
Here is the step-by-step guide to convert Sony HDR-SR10 videos to Flash on Mac.
Step 1. Load Sony HDR-SR10E videos to Aunsoft MTS/M2TS Converter for Mac.
Run Aunsoft MTS/M2TS Converter for Mac as the Mac Sony HDR-SR10 converter, and click the "Add" button to load .mts files for conversion.
Step 2. Choose Flash FLV output format.
Click the "Format" option, and choose Flash Video > FLV-Flash Video Format(*.flv) as output format. You can also choose the profiles like FLV H.264(*.flv) or F4V(/f4v).
With The above mentioned output, you shall use Flash player like JW player, Moyea PlayerBox player, or Flow player to play the Flash videos. If you want the video to play automatically, you may choose SWF-SWF Format(*.flv) as output.
Step 3. Transcode Sony HDR-SR10 MTS to Flash.
Click the "Convert" button under the preview window, and the conversion from Sony MTS to Flash video FLV/SWF will start right away.
Wait for a while, and you can upload the Flash videos to your sever and embed them on your web page.
1. The "Merge into one file" on the main interface is to join/splice .mts files.
2. If you are using camcorders like Sony HDR-CX350, HDR-XR520, HDR-TG7, or Cyber-shot DSC-TX7, DSC-H5V, you can also use the Mac Sony MTS converter to export FLV files for web sharing. Aunsoft MTS/M2TS Converter for Mac also works with Canon HF S21, Canon HF100, Panasonic HDC-TM700 and other AVCHD camcorders.


text:
Iris Van Riet, née le  est une femme politique belge flamande, membre de VLD, sénateur.

Elle est licenciée en sciences politiques et sociales (RUG);
directeur du Vlaams Centrum voor Volksontwikkeling.

Fonctions politiques
1999-2003: sénateur élu direct, en suppléance à Guy Verhofstadt, premier ministre.

Personnalité du Vlaamse Liberalen en Democraten
Sénateur belge
Naissance en avril 1952

redpajama_set_name: RedPajamaWikipedia


"Shhh." Eddie Mahone waved his free hand. His other gripped a fishing rod. With his one good eye, the detective surveyed the lake's calm, muddy surface.
For today's Daily Prompt, "Entertain," Wallie and I offer another snippet from a work-in-progress–a story in which a little girl is helped solving a crime by the ghost of a detective. The ghost is summoned to help her when Maddie plays a harp she found in the attic room. This is a scene in which he explains the harp to her.
'Do you want to know the story of my harp?' he asked.
I couldn't care less. But it was his delicate asking, and the way he pulled the chair to my bedside, that interested me.


When the season changes, I'm still stricken by the wardrobe. Do not assume an excessive amount of at this point, what's missing may be a sweater cardigan! I accustomed assume that it had been an easy grandparent, however the road individuals of the tides told you that the door to the new world is quietly gap.
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The fabric ink is used with Silhouette stencil vinyl by applying custom designs to apparel and fabric. It is soft-drying ink that better resists cracking and peeling. One bottle of ink is enough to create five to ten projects depending on size and intricacy. Inks are mixable to create custom colors.


Carrickduff National School in Carrickduff, Co. Carlow.
Map, location, contact details, directions to Carrickduff National School, Carrickduff, Co. Carlow. Add to your smart phone, Garmin or TomTom sat-nav. Find other nearby Schools.


HOLO/OR offers Diffraction gratings that are Binary Phase Gratings. These are a special case of a 1D Damman grating with a duty cycle of 50% within a period. Our gratings are transmission gratings, but can be offered in reflective versions as desired . In this section, we present our standard list of gratings with custom etching depth (modulation depth).
In addition to standard gratings, HOLO/OR can offer gratings with custom specifications (duty cycle, modulation depth, period, reflection or transmission grating).


I mentioned once before that I, too, was a victim of the Great Depression -- not that I went through it personally (I'm not THAT old), but I was raised by parents who did. The suffering continued.
Alice and Mitch's ultra-conservative approach to buying, using and disposing of consumables has stuck with me to this day. I am tortured by excess: Do I really need this extra lemon? Do I remember how to darn socks? Can't I wrap that gift in the cat food bag?
I have horrified my own children with a very cynical approach to food safety in general and expiration dates in particular: "They just put that there to sell more yogurt." I consume every single part of a piece of fruit, down to the stem and seeds.
In short, I really don't belong in America. Especially not in Texas, where we like to keep our larders full. When we say "conservative," we mean politics, not purchasing patterns.
As time goes by, the world is discovering the wisdom of the Alice & Mitch lifestyle. If we don't want to be pitching tents on top of landfills in the future, or lurching through smoke-filled megalopolises like Will Smith and Wall-E, we might want to take heed.
I started a list of tips for green living in September . But since then I've thought of a few more steps that were (a) maddeningly old fashioned to a wannabe hipster like myself, and (b) based on the best sort of eco-consciousness.
Welcome to going green, old school, part deux!
Anything other than soap is hogwash. The 1960's were the boom days for Proctor and Gamble, Betty Crocker, Kraft and Johnson & Johnson. Most of us were ready and willing to buy all the little conveniences like instant breakfast, spray-on furniture polish, plastic bags, and press-on nails.
Alice & Mitch would have none of it. My mother used Ivory soap to wash her floors and vinegar to wash the windows until 1995. She believed then, and I believe now, that anything you put on a surface that doesn't get rinsed off actually attracts dirt.
She saved a fortune on Windex and floor cleaner, and all those other concoctions that are living under my sink. And her house, in her heyday, was as clean as it gets.
If you're not using it, out it goes. My parents never collected much in the way of worldly goods. But what they did have got used -- or it got gone.
I was bemused to read that a principle of feng shui states that unused objects collect negative energy (which in Alice's world meant dust). Now, we all know what happens when the sorting and sifting, the passing along and the reassigning of material goods stops.
The @#$@# piles up. The stacks get higher, the closets fuller.
No one talks about how much energy it takes to keep your worldly goods in check. This is one of those life lessons I'm just learning now -- The less I bring in, the fewer decisions I have to make, the fewer "What the heck is this now?" looks I get from my kids, and the easier goes my life.
So this is my new version of saving energy. What's yours?
People think cloth napkins are only for company, but they're not. They're really a very practical, very economical way to keep yourself tidy, and save on paper napkins and towels in the mix. What's more, they can be beautiful, and much more useful than the flyaway things in the grocery store. If you're fond of eating outside, no need to anchor.
1. Get a few sets of napkins. For every day, you'll want a color that won't show every smudge and smear (but honestly, they don't get as gross as you would imagine) • 2. Give each person in your family a napkin they can call their own, at least for the next 2-3 meals • 3. After eating, fold and store where their rightful owners can find them -- or use individual napkin rings to distinguish. Each child could create his/her own? • 4. Wash with the rest of the kitchen linens. And ironing? Only for neatniks -- not a BH best practice. At all.
Beyond Her Napkins are a great way to go green. Colors and patterns vary, depending on where we source our blanks and what images we're fond of at the time. See them all on the Beyond Her website.


What really helped me improve my typing speed is...accuracy should come first, then speed. If you type correctly, muscle memory will kick in.
What really helped me improve my typing speed is...start slow, speed up as I go.
When my fingers hurt after too much time on the keyboard, I... do finger exercises and stretches.


Your role would be to clean and service bedrooms, bathrooms and corridors, as per company standard.
To clean and service bedrooms, bathrooms and corridors, as per company standard. Provide an efficient and personalised service and assist with new starters.
Work to room checklist as directed by Head Housekeeper.
Check duties and room allocation and collect tidies and trolley.
Clean and service bedrooms and bathrooms as per S.O.P., using guest's name on any occasion that you come into contact with them.
Ensure trolley and trolley bases are kept clean and tidy.
Requisition and control supplies of cleaning materials.
Ensure the security of guests' and company's property, stock and premises.
Report any necessary maintenance work.
Hand in completed comment cards.
Adhere to all regulations in respect of Health & Safety, hygiene, guest safety, fire regulations, emergency procedures, etc.
To comply with any reasonable request by the Head Housekeeper or Management.
Please email us at personnel@whittleburyhall.co.uk with your CV or application form. If you would like further information on this position, please call us on 01327 850 448.


LIMITED WARRANTY - Razor Golf Inc.
Subject to the EXCLUSIONS and OBLIGATIONS below, Razor Golf, Inc. warrants this product against defects in materials and workmanship for a period of one (1) year from the date of receipt. Razor Golf, Inc. is the sole warrantor of this product. Razor Golf, Inc. will replace the product at no charge if it fails to conform to this warranty. If the product has been discontinued, Razor Golf, Inc. reserves the right to replace it with a similar product of equal or greater value.
EXCLUSIONS: This warranty is effective only in the United States of America. It does not include product defects/conditions caused by abuse, alteration, mishandling, accident, commercial use or if the product has been serviced or repaired by someone other than the warrantor.
REPLACEMENT AS PROVIDED UNDER THIS WARRANTY IS THE EXCLUSIVE REMEDY AVAILABLE TO THE CONSUMER. RAZOR GOLF, INC. SHALL NOT BE LIABLE FOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES FOR BREACH OF ANY EXPRESS OR IMPLIED WARRANTY OF ITS PRODUCT(S). EXCEPT TO THE EXTENT PROHIBITED BY APPLICABLE LAW. ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IS LIMITED IN DURATION TO THE DURATION OF THIS WARRANTY.
Some states do not allow limitations on how long an implied warranty lasts and/or do not allow the exclusion of consequential damages, so the above limitations do not apply to those states. This warranty gives you specific legal rights. You may have other rights, which vary from state to state.
OBLIGATIONS: To obtain warranty service, the product must be delivered, at the owner's expense, (freight or postage paid) to the WARRANTOR with a written explanation of the reason for the return.


text:
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Tea flows as TCH Women's Club celebrates 25th anniversary
The Community House Women's Club celebrated its 25th anniversary with a Founders' Day Tea.
Tea flows as TCH Women's Club celebrates 25th anniversary The Community House Women's Club celebrated its 25th anniversary with a Founders' Day Tea. Check out this story on HometownLife.com: https://www.hometownlife.com/story/news/local/birmingham/2018/05/18/tea-flows-tch-womens-club-celebrates-25th-anniversary/597244002/
DIane K. Bert, Special to hometownlife.com Published 11:12 a.m. ET May 18, 2018
Ann Duncan (left), president of The Community House Women's Club, and historian Marion Beck celebrate the group's 25th anniversary. Beck has kept albums containing details and photos of activities over the years.(Photo: DIane K. Bert)
Twenty-five years!
Members of The Community House Women's Club recently hosted a Founders' Day Tea to celebrate the group's 25th anniversary. Some members wore fancy hats for the occasion.
Club president Ann Duncan welcomed the 77 guests. She describes the women as a group of professionals who enjoy each other, have a great lunch together and listen to a fine, thought-provoking speaker.
The club began 25 years ago, after many women requested a group similar to the Senior Men's Club. Some local women were envious of their husbands' social contacts and fun each Friday at the Men's Club. Meetings were held in 1992 and 1993 to assess interest.
A Birmingham Eccentric article from December 1992 stated, "So many people are affiliated with The Community House, it is their home away from home."
The response was enthusiastic and the club began its current pattern of meeting twice monthly, having a speaker, then dining. Interest groups formed as well. Currently, they include a book discussion group, a happy hour group, a movie group and a theater group. Ava Wixted helps organize the interest groups.
Marion Beck has served as historian of the group. Her beautifully made albums document its activities through the years.
"Our club has become very popular — some women are driving an hour to attend our luncheons and hear our speakers," said Maxine Smiley, who was a longtime editor of Vision, the club's quarterly newsletter.
At the celebration tea, tables were set with beautiful linens. A geranium plant with a butterfly was the centerpiece. Scones were the first course, followed by tea sandwiches and three dessert bon bons. The servers were conscientious about serving tea and refilling cups.
Animated conversations filled the ballroom at The Community House as members reunited and shared news. This is a typical scenario. Yo Karcher enjoys the conversations and getting out of the house. Pat Lodish especially enjoys the speakers.
Ann Duncan introduced past presidents of club and they received a hearty round of applause. These women included Carol Aubrey, Ava Wixted, Maxine Smiley, Helen Winslow, Helen Cost, Dee Keller, Leona Gates, Mary O'Neill and Beckie Perry. They each received a gift bag.
"It was a lot of work, but I enjoyed doing it," said Mary Ann Smyth, who co-chaired the event with her daughter Kim Daniels. "We had a fine committee."
O'Neill said she's been "a loyal member" for about 15 years.
"I enjoy the programs and everything about the group," she said. "It is on my calendar every other week."
Upcoming programs include Paul Ritchie, songwriter and entertainer; Barry King, talking about the Oakland County Child Killer Case; and meteorologist Paul Gross, talking about global warming. Joni Keller-Herman arranges programs.
To learn more and to make a luncheon reservation, call Lois Time at 313-806-2997.
The Community House Women's Club welcomes new members. Collette Pariseau is the membership chairman. She can be reached at 248-895-7282.
Diane K. Bert is a Bloomfield Hills resident with a Ph.D. in educational psychology from Wayne State University. She can be reached at dkbert617@gmail.com.
Read or Share this story: https://www.hometownlife.com/story/news/local/birmingham/2018/05/18/tea-flows-tch-womens-club-celebrates-25th-anniversary/597244002/
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