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Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya
Anne Kelly Kambura1,
Romano Kachiuru Mwirichia2,
Remmy Wekesa Kasili1,
Edward Nderitu Karanja3,
Huxley Mae Makonde4 &
Hamadi Iddi Boga5
BMC Microbiology volume 16, Article number: 136 (2016) Cite this article
Lake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi.
Water, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi "Nasikie eng'ida" (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 – V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package.
Three thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33–50 %), Firmicutes 3.52–28.92 %, Bacteroidetes (3.45–26.44 %), Actinobacteria (0.98–28.57 %) and Euryarchaeota (3.55–34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81–83 °C).
There were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.
Extreme environment refers to any setting that exhibits life conditions detrimental to living organisms with respect to its physicochemical properties such as pH, temperature, pressure, nutrient and saline concentration [1]. Extreme physicochemical parameters include acidity (pH <5), alkalinity (pH >9), hyper salinity (salinity >35 %), pressure (>0.1 MPa), high temperature (>40 °C), low temperature (<5 °C), water stress (aw <0.80), and high-radiation environments [2]. The extreme environments are inhabited by organisms referred to as extremophiles that are so well-adapted that they readily grow and multiply [3]. In Kenya, the haloalkaline soda lakes are characterized by exceptionally rich productivity rates presumably because of the high ambient temperatures, high light intensities, availability of phosphates and unlimited access to CO2 in these carbonate rich waters [4, 5]. Salinity levels can be as high as 30 % to saturation in Lake Magadi, whereas the pH ranges between 9 and 11.5 [6]. In Lake Magadi, solutes are supplied mainly by a series of alkaline springs with temperatures varying from 33 °C to 86 °C [6, 7].
Previous culture dependent and culture independent studies on Lake Magadi have revealed a dense and diverse population of aerobic, organotropic, halophilic, alkaliphilic, and haloalkaliphilic and alkalitolerant representatives of major bacterial phyla [8–15]. Although conventional microbial cultivation methods have helped shape understanding of physiology and metabolic functions of diverse organisms, they are laborious, time consuming, selective and biased for specific microbial growth. On the other hand, culture - independent studies done on soda lakes in Kenya have been based on the analysis of clone libraries of PCR amplified rDNA. This may not represent an accurate picture of prokaryotic diversity within a given community due to low speed and coverage of a cloning and Sanger-sequencing based approach, which gives a lower number of amplicon sequences compared to the millions of generated by High Throughput Sequencing technologies such as Illumina Sequencing [16]. This is the first culture independent study of the microbial community within the hot springs located around the hypersaline Lakes Magadi and Little Magadi. This study employed Illumina Sequencing of PCR products of both 16S rDNA and 16S rRNA cDNA to obtain a less biased estimation of microbial community within the hot springs' ecosystem. The main objective of this study was to analyze the targeted total community rDNA and cDNA generated from rRNA so as to compare the total versus active microbial communities within the hot springs of Lake Magadi and Little Magadi in Kenya.
Research authorization
Research authorization was obtained from National Commission for Science, Technology and Innovation (NACOSTI) on 30th August 2013 in Kenya, and permission to conduct research in Lake Magadi was obtained from Kenya Wildlife Services (KWS) on 24th September 2013.
Lake Magadi is a hypersaline lake that lies in a naturally formed closed lake basin within the Southern part of the Kenyan Rift Valley. It is approximately 2° 00′ 0″ S and 36° 00′ 0″ E of the Equator at an elevation of about 600 m above sea level [17]. The solutes are supplied mainly by a series of alkaline springs with temperatures as high as 86 °C which are located around the perimeter of the lake. Samples analyzed in this study were collected from three hot springs: one hot spring within the main L. Magadi (02° 00′ 3.7″S 36° 14′ 32″ E) at 45.1 °C and pH 9.8; and two hot springs within Little Magadi "Nasikie eng'ida" (01° 43′ 28″S 36° 16′ 21″E), and (01° 43′ 56″ S 36° 17′ 11″ E) at elevations of 611 and 616 m, temperatures of 81 and 83.6 °C and pH range of 9.2 and 9.4 respectively (Table 1).
Table 1 Parameters of sampling sites analyzed in this study
Measurements of physicochemical parameters
Geographical position of each site in terms of latitude, longitude and elevation was taken using Global Positioning System (GARMIN eTrex 20). The pH for each sampling point was measured with a portable pH-meter (Oakton pH 110, Eutech Instruments Pty. Ltd) and confirmed with indicator strips (Merck, range 5–10). Temperature, Electrical Conductivity (EC), Total dissolved solids (TDS) and dissolved oxygen (DO) were measured on site using Electrical Chemical Analyzer (Jenway - 3405) during sampling. In situ temperature was recorded once for each study site and assigned to all the three sample types for that site.
All samples were collected randomly in triplicates from each hot spring. Water samples were collected using sterile 500 ml plastic containers that had been cleaned with 20 % sodium hypochlorite and UV-sterilized for one hour. Wet sediments were collected by scooping with sterilized hand shovel into sterile 50 ml Falcon tubes. The upper 5 mm from each microbial mat developing on the hot spring water margins was collected into sterile 500 ml plastic jam jars. All samples were preserved on dry ice immediately after sampling, and transported to the laboratory in Jomo Kenyatta University of Agriculture and Technology. Water for nucleic acid extraction (500 ml) was filtered through a 0.22 μM filter membrane (Whatman) and all filter papers containing samples were stored at −80 °C. Pellets were obtained from water samples by re-suspending the filter papers in phosphate buffer solution, and centrifuging 5 ml of the suspension at 13,000 rpm for 10 min. These were used for nucleic acid extraction.
Nucleic acid extraction
Total community DNA was extracted from all the samples in triplicates; pellets from water samples, 0.2 g of sediment samples and 0.4 g of microbial mat samples, as described by as described by Sambrook et al. [18]. Total RNA was extracted from 0.25 g of sediment and mat samples, and pellets obtained from the water samples (described above), in triplicates using Trizol RNA extraction protocol [19]. The respective nucleic acids extracted from triplicate samples were pooled during the precipitation stage, the pellets were air dried and stored at −20 °C. The pellets were lyophilized to protect them from degradation.
Synthesis of cDNA from 16S rRNA
cDNA synthesis, amplification and sequencing were performed at Molecular Research DNA Lab (www.mrdnalab.com, Shallowater, TX, USA). The quality of total RNA was assessed using gel electrophoresis. The extracted RNA was dissolved in RNase-free water and subsequently treated to remove DNA contaminants using the Amplification Grade DNase I Kit (Sigma, MO) according to manufacturer's instructions. cDNA first-strand and second-strand synthesis was done using the Superscript III First-Strand Synthesis SuperMix (Invitrogen, CA) and the Second-strand cDNA Synthesis Kit (BeyoTime, Jiangsu, China), respectively, following manufacturer's instructions. Single-strand reverse transcription was done to provide template for amplicon libraries using Superscript III (Invitrogen) according to the manufacturer's protocol, random hexamer primed and with subsequent RNAse H digestion. The Double stranded cDNA synthesis was carried out as described by as described by Urich et al. [20].
Amplicon library preparation and sequencing
PCR amplification of the 16S rRNA gene V4 variable region was carried out from extracted DNA and cDNA generated from rRNA, using bacteria/archaeal primers 515 F (GTGCCAGCMGCCGCGGTAA) that had barcode and 806R (GGACTACHVGGGTWTCTAAT) according to Caporaso et al. [21]. Amplification proceeded in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) with initial denaturation heating at 94 °C for 3 min, followed by 28 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 40 s and extension at 72 °C for 1 min, and a final elongation at 72 °C for 5 min. The quality of PCR products was assessed on 2 % agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples, tagged with different barcodes, were pooled in equimolar ratios based on their DNA concentrations from the gel images. Pooled samples were purified using calibrated Ampure XP beads (Beckman Coulter) for use in library preparation. The pooled and purified PCR products were used to prepare 16S rDNA and cDNA library by following Illumina TruSeq DNA library preparation protocol [22]. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq 2x300bp Version 3 following the manufacturer's guidelines.
Sequence analysis, taxonomic classification and data submission
Sequences obtained from the Illumina sequencing platform were depleted of barcodes and primers using a proprietary pipeline (www.mrdnalab.com, MR DNA, Shallowater, TX) developed at the service provider's laboratory. Low quality sequences were identified by denoising and filtered out of the dataset [23]. Sequences which were <200 base pairs after phred20- based quality trimming, sequences with ambiguous base calls, and those with homopolymer runs exceeding 6 bp were removed. Sequences were analyzed by a script optimized for high-throughput data to identify potential chimeras in the sequence files, and all definite chimeras were depleted as described previously [24]. All this data filtering was done by the service provider using their pipeline. De novo OTU clustering was done with standard UCLUST method using the default settings as implemented in QIIME pipeline Version 1.8.0 at 97 % similarity level. Taxonomy was assigned to each OTU using BLASTn against SILVA SSU Reference 119 database at default e-value threshold of 0.001 in QIIME [25, 26]. Obtained sequences were submitted to the NCBI Sequence Read Archive with SRP# Study accessions: SRP061805. These included SRX1124606: RNA-Seq of Prokaryotes: Alkaline Hot springs and SRX1124607: DNA-Seq of Prokaryotes: Alkaline Hot springs (Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4).
Diversity indices (Shannon, Simpson and Evenness) for each sample were calculated using vegan package version 1.16-32 in R software version 3.1.3 [27]. Community and Environmental distances were compared using Analysis of similarity (ANOSIM) test, based upon Bray-Curtis distance measurements with 999 permutations. Significance was determined at 95 % confidence interval (p = 0.05). Calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS), redundancy analysis (RDA) and parameter correlation were carried out using the R programming language [27] and the Vegan package [28]. To support OTU-based analysis, taxonomic groups were derived from the number of reads assigned to each taxon at all ranks from domain to genus using the taxa_summary.txt output from QIIME pipeline Version 1.8.0.
Three hot springs of Lake Magadi and Little Magadi were selected based on different temperature and pH levels. Temperatures ranged from 45.1 to 83.6 °C while pH ranged from 9.2 to 9.8. The TDS was above the measurement range for the Electrical Chemical Analyzer; hence all the readings appeared as one (1). The metadata collected before sampling is summarized in Table 1.
Composition and diversity of the microbial communities
After denoising and demultiplexing, a total of 271,345 and 214,663 sequence reads were generated from 16S rDNA and 16S rRNA cDNA data respectively. Total OTU richness at 3 % distance amounted to 3502 and 1915 OTUs respectively. 85 and 62 OTUs were shared across all hot springs while 82 and 45 OTUs were shared across all sample types in the two data sets respectively. Figure 1a and b shows the distribution of OTUs across hot springs and sample types. For 16S rDNA data, hot spring 83.6 °C and 45.1 °C showed a relatively larger overlap (142 OTUs) than other hot springs, while 45.1 °C harbored most OTUs unique to one hot spring in both datasets.
a and b Venn diagrams showing the distribution of unique and shared OTUs within various sample types in the three sampling sites. The number of OTUs in each hot spring is indicated in the respective circle. Site A, B and C represent hot springs 83.6 °C, 81 °C and 45.1 °C respectively
Bacterial taxonomic composition analysis
16S rDNA OTUs comprised of Bacteria (85.55 %) and Archaea (11.27 %) while 16S rRNA cDNA OTUs comprised of Bacteria (83.22 %) and Archaea (13.95 %). These results suggest that bacteria are the most dominant taxa in L. Magadi and Little Magadi hot springs. The groups with highest relative abundances at phylum level belonged to members of Proteobacteria (16–31.4 %), Firmicutes (2.8–28.9 %), Bacteroidetes (3.4–23.8 %), Actinobacteria (1.0–14.4 %), Cyanobacteria (1.1–8.3 %, Chloroflexi (0.3–7.2 %), and Deinococcus-thermus (0.3–5.3 %) (Fig. 2). Other groups scoring high relative abundance in some samples include Gemmatimonadetes (24.1 %) in water samples at 81 °C, Planctomycetes (15.9 %) in water samples at 83.6 °C, Lentisphaerae 5.3 % in mat samples at 81 °C, Spirochaetae 4.5 % in wet sediment samples at 45.1 °C, Thermotogae (1.7 %) in wet sediment samples at 83.6 °C and Verumicrobia (3.9 %) in mat samples at 45.1 °C (Additional file 5: Table S5).
Relative abundance of the most predominant phyla in various samples collected from the hot springs of L. Magadi and Little Magadi. 'Others' represent all taxa that scored a relative abundance of below 1 % across all samples in both data sets
At family level, OTUs were distributed in 292 bacterial families with the most abundant belonging to Rhodobacteraceae that scored up to 9.43 % relative abundance in wet sediment samples at 45.1 °C, Cyanobacteria Family I (5.89 %), Desulfonatronaceae (4.55 %), Thermaceae (4.01 %), Ectothiorhodospiraceae (3.71 %), Spirochaetaceae (3.34 %), Nitriliruptoraceae (3.19 %), Anaerolineaceae (3.03 %), Peptococcaceae (3.03 %) and uncultured gamma proteobacterium (3.03 %) in various samples as shown in Additional file 6: Table S6.
Differences in relative abundance were seen as a function of sample type and temperature, with wet sediments harboring the highest taxa. The dominant taxa corresponded with those reported in previous studies conducted on deep sea and marine sediments community composition [29–31]. For example, a review by Brown et al. [30] on microbial life in extreme environments that compared metagenome analyses of different high thermal habitats, observed that microbes adapted to these habitats, are different with respect to species abundance and community structure. However, some bacterial taxa such as Thermotoga, Deinococcus-Thermus and Proteobacteria, were common within the samples under review [30]. These bacterial taxa were also found within the samples from the hot springs of Lakes Magadi and Little Magadi. Species diversity in high temperature environments has been shown to be relatively low [32, 33]. The deep-sea hydrothermal vent chimneys have been found to harbor Proteobacteria [34, 35], Bacteroidetes and Planctomycetes [36].
Archaeal taxonomic composition analysis
The OTUs were distributed among three Archaeal phyla; Euryarchaeota (1.76–34.48 %) across all samples, Crenarchaeota (up to 2.46 %) and Thaumarchaeota (up to 2.78 %) in wet sediment samples at 81 °C (Figs. 2 and 3). At the family level, OTUs were distributed in 24 families with the most abundant belonging to Halobacteriaceae (27.06 %) in water samples at 81 °C, Deep Sea Hydrothermal Vent Gp 6 (DHVEG-6) (0.35–5.15 %) across all samples, South African Goldmine Gp (SAGMEG); Uncultured Archaeon (4.01 %) and Methanocaldococcaceae (2.47 %) in wet sediment samples at 81 °C (Additional file 6: Table S6). Crenarchaeota phyla members identified belonged to the families Desulfurococcaceae (0.93 %), Thermoproteaceae (0.93 %), Thermofilaceae (0.61 %) in wet sediment samples at 81 °C and Sulfolobaceae (0.42 %) in wet sediment samples at 83.6 °C, while Thaumarchaeota were mainly assigned to uncultured archaeon with up to 1.68 % relative abundance in wet sediment samples at 83.6 °C (Additional file 6: Table S6). Previous studies on thermal groundwater in a thermal field in Russia showed that Archaea is dominated by a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales (39 % of all archaea) and by another abundant group (33 % of all archaea) related to the phylum Crenarchaeota. Both groups are widely spread in hot springs all over the world [37]. Some Archaeal taxa such as Methanococcus, Thermoprotei and Thermococcus were also common within the samples under review by Brown et al. [30]. These are similar to the classes obtained in this study, indicating that Archaea are well adapted to extreme conditions and could be responsible for various functional processes within the ecosystem.
Relative abundance of the most abundant prokaryotic taxa at order level in samples from the hot springs of L. Magadi and Little Magadi. 'Others' represent all taxa that scored a relative abundance of below 1 % across all samples in both data sets
Active diversity taxonomic composition based 16S rRNA cDNA analysis
Bacterial taxonomic composition
The 1913 OTUs of 16S rRNA cDNA data set were dominated by bacterial phyla comprising Proteobacteria (8.3–50 %) and Actinobacteria (2.0–28.6 %) across all samples. The data confirm the predominance of previously defined groups belonging to Proteobacteria [38]. Other groups scoring high relative abundance in some samples include Bacteroidetes (26.4 % in wet sediment samples at 45.1 °C), Chloroflexi (5.3 % in water samples at 45.1 °C), Firmicutes (23.9 % in mat samples at 45.1 °C), Cyanobacteria (8 % in mat samples at 45.1 °C), Gemmatimonadetes (25 % in water samples at 81 °C), Lentisphaerae (7.1 % in wet sediment samples at 83.6 °C) and Planctomycetes (6.6 % in mat samples at 83.6 °C) (Fig. 2).
Unlike 16S rDNA dataset, the most abundant bacterial families found within cDNA dataset belonged to Methylophilaceae; scoring up to 8.3 % relative abundance in some samples, Corynebacteriaceae, Dermatophilaceae, Micrococcaceae, Nocardioidaceae, Bacteroidaceae; ML635J-40 aquatic group, Sphingomonadaceae, Burkholderiaceae, and Myxococcales; 0319-6G20, Alcanivoracaceae, Pseudomonadaceae (7.14 %), Desulfuromonadales; GR-WP33-58 (5.11 %), Cyanobacteria; Subsection III; Family I, Syntrophomonadaceae (4.7 %), Spirochaetaceae (3.7 %), Anaerolineaceae (3.44 %), Gemmatimonadetes; BD2-11 terrestrial group; uncultured bacterium 3.3 %, Rhodospirillaceae (3.3 %) and Lentisphaerae (ML1228J-2; uncultured bacterium) (2.6 %) (Additional file 7: Table S7).
Archaeal taxonomic composition
16S rRNA cDNA OTUs were distributed among three Archaeal phyla, similar to those obtained from the 16S rDNA dataset. These were Euryarchaeota (6.6–28.4 %) across all samples, Crenarchaeota (up to 1.38 %) in water samples at 81 °C and Thaumarchaeota (up to 1.15 %) in wet sediment samples at 45.1 °C (Fig. 3). The most abundant families belonged to Halobacteriaceae (21.31 %), Deep Sea Hydrothermal Vent Gp 6 (DHVEG-6) (8.59 %), and Marine Benthic Group D and DHVEG-1 (2.29 %). Crenarchaeota phyla members identified belonged to the families Desulfurococcaceae (1.11 %) in wet sediment samples at 81 °C and Sulfolobaceae (0.28 %) in wet water samples at 45.1 °C, while Thaumarchaeota were mainly assigned to uncultured archaeon with up to 1.15 % relative abundance in wet sediment samples at 45.1 °C (Additional file 7: Table S7). Previously, several 16S rRNA gene sequences related to novel Archaea (Euryarchaeota) were retrieved from the alkaline saltern at Lake Magadi [13]. Haloalkaliphilic Archaea related to Natronomonas, Natrialba, Natronolimnobius and Halorubrum spp. have also been isolated from Lake Magadi and Inner Mongolian soda lakes [39].
Microbial richness and diversity indices
Using rarefaction, the same number of sequences from each sample was used in comparison of community alpha and beta diversity measures. Paired t-tests at class taxonomic level of both 16S rDNA and 16 rRNA cDNA indicated that significant differences between samples based on alpha diversity indices whose values obtained were as follows: Shannon diversity index (H'); wet sediment 83.6 °C (7.9 vs 3.8), water 81 °C (9.1 vs 3.6) and Simpson (1/D); wet sediment 83.6 °C (34.54 vs 12.2); water 81 °C (7.76 vs 6) and water 45.1 °C (28.53 vs 10.3) respectively (Table 2).
Table 2 Diversity indices computed on all OTU-based microbial taxonomic units within 16S rDNA and 16S rRNA cDNA datasets
Total microbial diversity based on 16S rDNA and 16S rRNA cDNA ANOSIM at order level showed that there were significant differences in microbial community structure in the samples at 95 % level of confidence (P value, 0.009), and 0.383 R statistic value while active microbial diversity based on 16S rRNA cDNA had (P value, 0.01), and 0.333 R statistic value. This could be attributed to differences in temperature and pH of the specific sites during sampling. Samples from 45.1 °C harbored more closely related populations because it had less extreme conditions as compared to the two other hot springs.
Distance based redundancy analysis showed that the microbial community evenness significantly differed from each site for both 16S rDNA and 16S rRNA cDNA. 16S rDNA ordination of the three sample types showed a significance of 0.017 and while 16S rRNA cDNA dataset showed a significance of 0. 011. Samples from each site clustered close to each other in separate quadrants indicating high beta diversity between the three sampling sites.
NMDS analyses supported by OTU and taxonomic composition, divide the datasets into three ellipses: one for each hot spring. Some microbial taxa were shared between habitats in both 16S rDNA and 16S rRNA cDNA derived datasets. This scenario was more pronounced in 16S rDNA - derived dataset, indicating that DNA pool contained a "seed bank" of inactive and sporulating organisms [40], while fewer taxa were active within the ecosystem as shown in the 16S rRNA cDNA derived dataset (Fig. 4). Similar results were observed in a study on Ethiopian soda lakes where NMDS analyses supported both by OTU and taxonomic composition; divided the datasets into six well-separated habitats with relatively few OTUs that were shared between more than one or two habitats [41]. The taxa were also observed to cluster according to sample types (i.e. wet sediments, microbial mats and water samples). There was an overlap of taxonomic clusters between wet sediments and microbial mats from the three sample types in both 16S rDNA and 16S rRNA cDNA derived datasets. However, water samples formed separate clusters from the other two sample types in both 16S rDNA and 16S rRNA cDNA datasets (Figs. 4 and 5).
a Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities between microbial compositions of 16S rDNAdataset, grouped according to sampling sites. Site A, B and C represent hot springs 83.6 ºC, 81 ºC and 45.1 ºC respectively. The boxes 1 - 3 represent wet sediments, mats and water samples from 83.6 ºC, 4 - 6 represent wet sediments, mats and water samples from 81 ºC and 7 - 9 represent wet sediments, mats and water samples from 45.1 ºC. b Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities between microbial compositions of 16S rRNA cDNAdatasets grouped according to sampling sites. Site A, B and C represent hot springs 83.6 ºC, 81 ºC and 45.1 ºC respectively. The boxes 1 - 3 represent wet sediments, mats and water samples from 83.6 ºC, 4 - 6 represent wet sediments, mats and water samples from 81 ºC and 7 - 9 represent wet sediments, mats and water samples from 45.1 ºC
a Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities between microbial compositions of 16S rDNAdatasetgrouped according to sample types. Each ellipse represents a set of the three sample types collected from each hot spring. The boxes 1 - 3 represent wet sediments, mats and water samples from 83.6 ºC, 4 - 6 represent wet sediments, mats and water samples from 81 ºC and 7 - 9 represent wet sediments, mats and water samples from 45.1 ºC. b Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities between microbial compositions of 16S rRNA cDNA dataset grouped according to sample types. Each ellipse represents a set of the three sample types collected from each hot spring. The boxes 1 - 3 represent wet sediments, mats and water samples from 83.6 ºC, 4 - 6 represent wet sediments, mats and water samples from 81 ºC and 7 - 9 represent wet sediments, mats and water samples from 45.1 ºC
Hierarchical clustering between samples collected from lake Magadi and Little Magadi revealed samples from the two hot springs in Little Magadi "Nasikie eng'ida" were closer than samples from the hot spring in the main lake (Fig. 6 (a and b) and (Additional file 8: Figure S1 and Additional file 9: Figure S2).
a Hierarchical clustering of 16S rDNA samples collected from the three hot springs of L. Magadi and Little Magadi. Phylum level was chosen to be used in hierarchical clustering to assess the relationships between samples and taxa. "Sed" represent wet sediment samples from respective temperature. b Hierarchical clustering of 16S rRNA cDNA samples collected from the three hot springs of L. Magadi and Little Magadi. Phylum level was chosen to be used in hierarchical clustering to assess the relationships between samples and taxa. "Sed" represent wet sediment samples from respective temperature
The combined findings of this study, show that estimated diversity and richness within the hot spring samples were found to be as high as those found in other environments such as soil and deep-sea hydrothermal environments [42]. The results confirm that different groups of microorganisms have the capacity to adapt and thrive even in the most hostile environments. Some of these groups (Acidobacteria; Blastocatella, Bryobacter and Telmatobacter genera, Bacteroidetes; Bacteroidales, Rhodothermaceae, Flavobacteriaceae, Sphingobacteriales and Chloroflexi; Dehalococcoidales and Thermomicrobia) also obtained from previous similar studies were reported to have a fermentative ability [41]. It was observed that from the cDNA dataset, photosynthetic taxa were represented by Cyanobacterial genera Leptolyngbya and Lyngbya, among other uncultured groups. Primary production within the hot springs is probably supported by some groups of non-sulfur purple bacteria from the family Rhodobacteraceae (specifically the genera Roseobacter that scored 1.1 % relative abundance), and purple sulfur bacteria from the family Ectothiorhodospiraceae present across samples at different relative abundance. The presence of Planctomycetes within samples could be an indicator that anaerobic ammonium oxidation may be another metabolic pathway supporting primary production in the low-oxygen, saline environment, since the dissolved oxygen concentration of the sampling sites ranged between 0.04 and 12.4 mg/l. Actinobacteria and Firmicutes are believed to have adaptive advantage under low-nutrient conditions of the highly alkaline, saline hot springs hence their high relative abundance levels. The presence of sulfate reducers in the family Desulfohalobiaceae (mainly Desulfonatronovibrio), suggested an internal sulfur cycle within the lake, as previously suggested for Khadin, Tuva, Russia and Natron soda lakes [42, 43]. Taxa typical for highly specialized metabolisms that were encountered in this study include Nitriliiruptor, known for their ability to catabolize nitriles or cyanides [44] and heterotrophic Oceanospirillaceae (Marinospirillum). Other functional taxa encountered include aerobic heterotrophs (e.g. Bacteroidetes, Marinicella) and fermentative anaerobes such as Thermoplasmatales among other uncultured groups. Euryarchaeota members were clustered into the classes, Halobacteria, Methanobacteria, Methanomicrobia, Methanococci, Thermococci and Thermoplasmata while Crenarchaeota phyla comprised Thermoprotei class. Previously, Methanobacteria and Methanomicrobia have been reported in oilfields, while Halobacteria and Thermoprotei have been reported in petroleum reservoirs [45]. Some Halobacteria members are important in organic fertilizer production industry as Lignin decomposers [46]. However, most of the genera identified in this study are known to be heterotrophs responsible for the primary degradation of organic matter [39]. The actual function of microbial taxa reported in this study could further be explored and established using culture dependent methods as well as mRNA transcripts.
In conclusion, this study presented microbial diversity analysis of samples collected from the hot springs of L. Magadi and Little Magadi based on both DNA and RNA, using Illumina Sequencing Technology. The results showed comparable profiles of microbial community using 16S rDNA and 16S rRNA cDNA derived datasets, hence indicating that the observed diversity is real. The findings showed a broad microbial distribution with water from the spring at 83.6 °C found to be the richest sample, constituting 680 observed species. Despite the fact that the sampling environment is multi-extreme due to high pH, temperature, and salinity, this study shows that there are stable and active microbial communities that have adapted to this environment. Culture dependent studies in future will help us unravel the survival mechanisms used by these polyextremophiles.
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We thank National Commission for Science, Technology and Innovation (NACOSTI) for providing a Research Grant to this work and Higher Education Loans Board (HELB) for providing a partial scholarship for school fees. We also thank Kenya Wildlife Services (KWS) and the NACOSTI provided permits for sample collection in Lake Magadi.
The Research was funded by the National Commission for Science, Technology and Innovation.
Availability of data and material
The data is available on NCBI Sequence Read Archive with SRP# Study accessions: SRP061805. SRX1124606: RNA-Seq of Prokaryotes: Alkaline Hot springs and SRX1124607: DNA-Seq of Prokaryotes: Alkaline Hot springs.
Conceived and designed the experiments: AKK EKN HIB RM. Performed the experiments: AKK EKN RWK RM. Analyzed the data: AKK EKN RM HMM. Contributed reagents/materials/sequencing costs/analysis tools: HIB EKN RM, RWK. Wrote the paper: AKK HIB RWK EKN HMM RM. All authors read and approved the final manuscript.
All authors have consented to the publication of this manuscript.
New clinical tools and procedures
Research was authorized by National Commission for Science, Technology and Innovation and Kenya Wildlife Services.
Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya
Anne Kelly Kambura & Remmy Wekesa Kasili
Embu University College, P. O. Box 6-60100, Embu, Kenya
Romano Kachiuru Mwirichia
International Centre of Insect Physiology and Ecology (ICIPE), P. O. Box 30772-00100, Nairobi, Kenya
Edward Nderitu Karanja
Pure and Applied Sciences, Technical University of Mombasa, P. O. Box 90420-80100, GPO, Mombasa, Kenya
Huxley Mae Makonde
Taita Taveta University College, P.O. Box 635-80300, Voi, Kenya
Hamadi Iddi Boga
Anne Kelly Kambura
Remmy Wekesa Kasili
Correspondence to Hamadi Iddi Boga.
Additional file 1: Table S1.
Total diversity environmental data sheet of the samples collected from the hot springs of L. Magadi and Little Magadi (XLS 25 kb)
Active diversity environmental data sheet of the samples collected from the hot springs of L. Magadi and Little Magadi (XLS 24 kb)
Total diversity Sequence Read Archive metadata sheet of the samples collected from the hot springs of L. Magadi and Little Magadi (XLSX 50 kb)
Active diversity Sequence Read Archive metadata sheet of the samples collected from the hot springs of L. Magadi and Little Magadi. (XLSX 50 kb)
Relative abundance of microbial taxa at phylum level in various samples collected from the three hot springs (83.6, 81 and 45.1 °C) of L. Magadi and Little Magadi. 'No blast hit/Others' represent unclassified sequences in both data sets. (XLSX 17 kb)
Total diversity relative abundance of microbial taxa at family level in various samples collected from the three hot springs (83.6, 81 and 45.1 °C) of L. Magadi and Little Magadi. 'No blast hit/Others' represent unclassified sequences in both data sets. (XLSX 45 kb)
Active diversity relative abundance of microbial taxa at family level in various samples collected from the three hot springs (83.6, 81 and 45.1 °C) of L. Magadi and Little Magadi. 'No blast hit/Others' represent unclassified sequences in both data sets. (XLSX 32 kb)
Additional file 8: Figure S1.
Comparative analysis (UPGMA similarity tree) of total microbial diversity of various sample types within hot springs of L. Magadi and Little Magadi. (DOCX 55 kb)
Comparative analysis (UPGMA similarity tree) of active microbial diversity of various sample types within hot springs of L. Magadi and Little Magadi. (DOCX 56 kb)
Kambura, A.K., Mwirichia, R.K., Kasili, R.W. et al. Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya. BMC Microbiol 16, 136 (2016). https://doi.org/10.1186/s12866-016-0748-x
Lake Magadi
16S rRNA cDNA
Microbial diversity


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Home » Theology » Soteriology & Sanctification
Balancing the Christian Life
For 25 years, people have looked to this book--which focuses on the Bible as the one, true guide to spirituality--for guidelines to genuine and wholesome living. In this special 25th anniversary edition, Moody Press commemorates the author and his contributions to the Christian community.
Charles C Ryrie
BECOMING THE GOSPEL PAUL PARTICIPATION & MISSION
The first detailed exegetical treatment of Paul's letters from the emerging discipline of missional hermeneutics, Michael Gorman's Becoming the Gospel argues that Paul's letters invite Christian communities both then and now to not merely believe the gospel but to become the gospel and, in doing so, to participate in the life and mission of God.
Showing that Pauline churches were active public participants in and witnesses to the gospel, Gorman reveals the missional significance of various themes in Paul's letters. He also identifies select contemporary examples of mission in the spirit of Paul, inviting all Christians to practice Paul-inspired imagination in their own contexts.
Michael J Gorman
William B. Eerdmans Publishing Company
Charity & Its Fruits
Spring 2020 Req. Dts
Sermons on 1 Corinthians 13 which give a rich insight into Edwards' regular pulpit ministry. The concluding chapter is on heaven as a world of love.
J Edwards
Christian Spirituality: The Classics is a unique and comprehensive guide to thirty key Christian spirituality texts. Ranging from Origen and Augustine to Jonathan Edwards, Thérèse of Lisieux and Thomas Merton, it offers a view of the texts which is founded in scholarship, but which also presents them as living documents that invite- even compel -contemplative reflection and existential response.
Each chapter briefly describes the classic text's author and audience, gives a synopsis of its contents, suggests some of its influence in history, and then explores aspects of the text's meaning for readers today. Key themes include:
How can human beings find truth?
How can they discover who they really are?
How can they live together in peace?
How can they live more fully in God's presence in this world and be united with God in the world to come?
The scholars who have written these chapters are all experts on their respective topics, but they wear their learning lightly. Anyone wishing to discover the riches of Christian spirituality will find this the ideal introduction and should be able to progress to a deeper understanding of the texts themselves.
COMMANDS OF CHRIST WHAT IT REALLY MEANS
Jesus says in John 15:10-11, "If you keep My commands you will remain in My love, just as I have kept My Father's commands and remain in His love. I have spoken these things to you so that My joy may be in you and your joy may be complete."
The Commands of Christ is an unpacking of that key promise, a simply powerful presentation of the loving imperatives we sometimes know more by rote than by heart: Love your enemies; Don't store up treasures on earth; Seek first the kingdom of God; Let your light shine before men, etc.
Tom Blackaby's focused writing serves as a plumb line of sorts, guiding us toward a vibrant commitment to the Lordship of Christ that better shapes our lives, how we worship, and how we relate to others. You will begin to know more clearly and experience more fully the freedom that comes through obedient acts of love and service to God.
Tom Blackaby
Community Called Atonement
Over the centuries the church developed a number of metaphors, such as penal substitution or the ransom theory, to speak about Christ's death on the cross and the theological concept of the atonement. Yet too often, says Scot McKnight, Christians have held to the supremacy of one metaphor over against the others, to their detriment. He argues instead that to plumb the rich theological depths of the atonement, we must consider all the metaphors of atonement and ask whether they each serve a larger purpose.
A Community Called Atonement is a constructive theology that not only values the church's atonement metaphors but also asserts that the atonement fundamentally shapes the life of the Christian and of the church. That is, Christ identifies with humans to call us into a community that reflects God's love (the church)--but that community then has the responsibility to offer God's love to others through missional practices of justice and fellowship, living out its life together as the story of God's reconciliation. Scot McKnight thus offers an accessible, thought-provoking theology of atonement that engages the concerns of those in the emerging church conversation and will be of interest to all those in the church and academy who are listening in.
DISTINGUISHING TRAITS OF CHRISTIAN CHARA
Gardiner Spring (February 24, 1785 - August 18, 1873) was an American minister and author.
Gardiner Spring
Northampton Press
Enemy Within Straight Talk about the Power & Defeat of Sin
Drawing from two masterful works by John Owen, Kris Lundgaard offers insight, encouragement, and hope for overcoming the enemy within.
Kris Lundgaard
P, R Publishing
Five Views on the Extent of the Atonement
For whom did Christ die? Who may be saved? are questions of perennial interest and importance for the Christian faith. In a familiar Counterpoints format, this book explores the question of the extent of Christ's atonement.
Zondervan Academic
FOR ALL THE SAINTS
How do theology and spirituality relate to one another? How does the Christian heart connect with the Christian mind? This collection of essays from leading evangelical theologians and writers addresses these concerns through providing scholarly and personal reflections. Here you will find discussion of the integration of theology and spirituality, biblical and classical sources for spiritual formation, a critique of how evangelicals have uncritically appropriated the rhetoric of spirituality, and also the use and abuse of spiritual disciplines by evangelicals.
Foundations of Spiritual Formation a Community Approach to Becoming Like Christ
Foundations of Spiritual Formation takes a unique approach to its subject, arguing that we become like Christ in the context of authentic, Christian community. Without undermining individual Bible study, private prayer, and meditation, the authors emphasize these pursuits for the purpose of both personal and community enrichment-that the whole body, as well as the individual, may be built up.
Part 1 lays the groundwork of spiritual formation. Jonathan Morrow develops a distinctively evangelical theology, while Richard Averbeck writes about worship. Then Gordon Johnston and Darrell Bock delve into the text of Scripture, grounding the pursuit of spiritual formation in revealed truth.
Part 2 focuses on functional aspects of spiritual formation. Klaus Issler emphasizes the importance of the heart in spiritual formation, while Reid Kisling illustrates the vital connection between character development and spiritual formation. Bill Miller explores love's role as the motivation for spiritual formation. Andrew Seidel examines servant leadership, and George Hillman extends the discussion to include the significance of calling. Gail Seidel discusses personal narrative as a catalyst for spiritual formation, and in closing, Harry Shields advocates the public preaching of the Word as a tool for spiritual formation.
Paul Pettit

redpajama_set_name: RedPajamaCommonCrawl


An inviting landscape is a wonderful way to make a first impression when you're welcoming guests into your home. If you're planning on putting your house on the market, it's also a smart way to add value to your property.
A well-kept entryway that is designed to work in harmony with your home's architectural elements will boost curb appeal and set the stage for a lovely visit.
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Be Practical: Every home presents some challenges that should be addressed head-on. For example, concealment plantings can be strategically placed to hide unsightly features such as foundation lines, HVAC systems or garbage collection areas. And in many homes, the front door is simply ornamental, as almost all guest traffic enters through a side door. By acknowledging traffic patterns around your home, we can create an inviting design that will either re-direct visitors to your front door, or create a landscape design that will elevate your side entrance to an inviting entryway you'll be proud of.
Whether you are simply freshening up your entryway, or you would like a complete redesign, we're here to help! The landscape design experts at Handy Andy Outdoors create customized designs that maximize our clients' return on investment and take their front entryway to the next level.


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magnetic treatment of irrigation water: its effects in brazil
Magnetic treatment of irrigation water: Its effects
The irrigation water of different types was treated with a magnetic device before applying to the plants. The mean values of pH, EC, N, P and K values of different irrigation water types before and after magnetic treatment are presented in Table 1.Magnetic treatment of water tends to reduce slightly the water pH, while there is no apparent trend for EC values.
In particular, the magnetic treatment of recycled water and 3000 ppm saline water respectively increased celery yield by 12% and 23% and water productivity by 12% and 24%. For snow peas, there were 7.8%, 5.9% and 6.0% increases in pod yield with magnetically treated potable water, recycled water and 1000 ppm saline water, respectively.
Magnetic treatment of irrigation water: Its effects on vegetable crop yield and water productivity Article (PDF Available) in Agricultural Water Management 96(8):1229-1236 · August 2009 with
Downloadable (with restrictions)! This study examines whether there are any beneficial effects of magnetic treatment of different irrigation water types on water productivity and yield of snow pea, celery and pea plants. Replicated pot experiments involving magnetically treated and non-magnetically treated potable water (tap water), recycled water and saline water (500ppm and 1000ppm NaCl for
Magnetic Treatment of Irrigation Water: Experimental
The effects of magnetic treatment on irrigation water have been studied. We showed that the main effects were the increase of the number of crystallization centers and the change of the free gas content. Both effects improve the quality of irrigation water. As an example, changes in natural water due to magnetic treatment in a commercially available apparatus, Magnalawn 2000, have been studied
Magnetic Treatment of Irrigation Water and its effect
causes re-alignment of water molecules which results in the alterations of water structure. In simple the treatment by magnetic field can bring about changes in density, surface tension, viscosity and other water properties [7]. Magnetic field is known to create the asymmetry of hydrated shells due to its effects on water molecules
The impacts of magnetic treatment of irrigation water
By keeping all these points, an attempt has been made to study the effects of magnetic treatment of irrigation water types viz., normal irrigation water, saline water and hard water and its influence on the growth and yield parameters on different crops; influence on soil moisture and water properties. 2. Materials and methods 2.1. Study site
Magnetic Water Technology | Soil Permeability | Reduce
Magnetic Treatment of Irrigation Water: Evaluation of its Effects on Vegetable Crop Yield and Water Productivity. Agricultural Water Management, 96(8): 1229-1236. Elsevier International Peer Review. Further controlled studies show the following results on varying crops:
Removal of Cryptosporidium by wastewater treatment
Cryptosporidium is a protozoan parasite that infects humans and various animal species. The environmental stability and the low infectious dose of Cryptosporidium facilitate its transmission by water and food. Discharge of untreated wastewater may result in waterborne or foodborne Cryptosporidium outbreaks, therefore a suitable treatment may prevent its dissemination.
Produced water treatment by nanofiltration
be used to the treatment of water with a concentration nearly 2000 mg.L-1, using a range of pressures from 2 until 25 bars (Alzahrani et al., 2013; Da Motta et al., 2013; Giacobbo et al., 2013). This work aims to evaluate the efficiency of the nanofiltration process in PW treatment for its reuse,
Water & Wastewater | Yokogawa Austria
The municipal water system of Oviedo, Florida provides high quality water to more than 30,000 area residents from its West Mitchell Hammock and A.M. Jones water treatment facilities. The West Mitchell Hammock facility is designed to provide an average daily flow of 10 million gallons per day (MGD), with an ultimate design flow of 16 MGD.
Alleviating the impact of water stress
irrigation at the semi-aerobic condition resulted in a 20% decline in its concentration, which helped boost growth and grain yield. The reverse phenomenon occurred in the con-centration of TSP while changing soil water content. Its concentration under the aerobic condition was 27% less than that in semi-aerobic conditions.
Climate Change Would Increase the Water Intensity
Changes in atmospheric CO2 concentrations, temperature, and precipitation affect plant growth and evapotranspiration. However, the interactive effects of these factors are relatively unexplored, and it is important to consider their combined effects at geographic and temporal scales that are relevant to policymaking. Accordingly, we estimate how climate change would affect water requirements
Chelating and antibacterial properties of chitosan
Chelating effect of chitosan nanoparticles. After the irrigation procedures, the samples were dried at room temperature for 24 hours and fixed on stubs. Three micrographs per block were analysed using a PSEM with a magnification of 500 times at 10 kV creating a total of 150 images. The area of each image represented 1,024 µm × 1,024 µm.
Co-Occurrence of Triclocarban and Triclosan in U.S. Water
Triclocarban (TCC) and triclosan (TCS) are antimicrobial additives in personal care products. Whereas TCS has been studied extensively, the environmental fate of TCC remains largely unknown. To address this data gap, we performed quantitative structure−activity relationship (QSAR) analyses that suggested a propensity of TCC to persist in various environmental compartments with predicted half
Nanotechnology for clean water: Facts and figures - SciDev.Net
Nanotechnology could help give millions clean drinking water. David J. Grimshaw outlines the potential, the progress and some of the risks.. Technology has long been important in providing clean drinking water and irrigation for food crops.Indeed, people have had water technology for thousands of years — the Romans were using aqueducts as conduits for drinking wateraround 300BC.
10 Interesting Articles About the Global Water Crisis
China's Looming Water Crisis The Ecologist "According to Jiang Liping, senior irrigation specialist at the World Bank in Beijing, China is over-exploiting its groundwater by 22 billion cubic meters a year – yet per capita water consumption is less than one third of the global average."
The Use of Subsurface Barriers in the Sustainable
of groundwater and provide storage for water investigators to determine quality variation and underground is a common practice in many parts of the determine heads and flows in the vicinity of the barrier. A world, notably in India, Africa, Brazil, Japan and geostatistical approach to the inverse problem of southwest of Saudi Arabia.
Condition monitoring and the effects of bearings on water
In order to keep reliability high, various condition monitoring/diagnostic tools should be employed for water pumps. The vibration, the wear, the flow, the pressure and the temperature are the important condition monitoring parameters/techniques to predict the health of any rotating machinery.
How to prevent solenoid valve noises | Tameson
How to prevent solenoid valve noises. They smoothen out the varying strength of the magnetic field by storing magnetic energy while the current is at its peak, and releasing it when the AC current is near to zero. In order to avoid or reduce these effects, pneumatic mufflers or silencers can be installed in exhaust ports.
CDC - Parasites - Water
When water becomes contaminated by parasites, however, it can cause a variety of illnesses. Globally, contaminated water is a serious problem that can cause severe pain, disability and even death. Common global water-related diseases caused by parasites include Guinea worm, schistosomiasis, amebiasis, cryptosporidiosis (Crypto), and giardiasis
AGRIS: International Information System for the Agricultural Science and Technology
Water purification is the process of removing undesirable chemicals, biological contaminants, suspended solids, and gases from water. The goal is to produce water fit for specific purposes. Most water is purified and disinfected for human consumption (drinking water), but water purification may also be carried out for a variety of other purposes, including medical, pharmacological, chemical
CIHEAM © CIHEAM-IAMB,
CIGR CIHEAM - IAMB Water, environment and agriculture: challenges for sustainable development CIGR Inter - Regional Conference on Land and Water Challenges 1st N. Lamaddalena, M. Todorovic, L.S. Pereira (Eds.)
Air and Water Pollution | List of High Impact Articles
Water treatment containing organic compounds by coupling adsorption and electrochemical degradation at BDD anode: Sawdust adsorption performance for the treatment of dilute phenol solutions PPT Version | PDF Version; Enrique M. Ostrea Alluvial and riparian soils as major sources of lead exposure in young children in the Philippines: The role of
Time-Dependent Effects of Chitosan on Dentin Structures
Time-dependent effects of chitosan 359 slight peritubular erosion in part of the dentinal tubules was verified (Fig. 1B). In G2, complete removal of the smear layer with all the tubule openings was clearly visible (Fig. 1C), without the presence of the smear plug, but with slight erosive effect on the peritubular dentin (Fig. 1D).
Manganese is a chemical element with the symbol Mn and atomic number 25. It is found as a free element in nature; it is often found in minerals in combination with iron.Manganese is a transition metal with a multifaceted array of industrial alloy uses, particularly in stainless steels.
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text:
/*
 * To change this license header, choose License Headers in Project Properties.
 * To change this template file, choose Tools | Templates
 * and open the template in the editor.
 */
package Controller;

import Model.AutomovelModel;
import java.util.ArrayList;
import valueObject.Automovel;

/**
 *
 * @author cristhian
 */
public class AutomovelController {

    public static void cadastrarAutomovel(Automovel automovel) {
        boolean verifica = AutomovelController.verificarCampos(automovel);
        if(!verifica) {
            automovel.setError(true);
            // Algum dado informado é inválido
            return;
        }
        AutomovelModel.cadastrarAutomovel(automovel);
    }
    public static void alterarAutomovel(Automovel automovel) {
        boolean verifica = AutomovelController.verificarCampos(automovel);
        if(!verifica) {
            automovel.setError(true);
            // Algum dado informado é inválido
            return;
        }
        AutomovelModel.alterarAutomovel(automovel);
    }
    public static void excluirAutomovel(Automovel automovel) {
        AutomovelModel.excluirAutomovel(automovel);
    }

    private static boolean verificarCampos(Automovel automovel) {
        String mensagem = "";

        // renavam
        if(!automovel.getRenavam().matches("[0-9]{11}"))
            mensagem = mensagem.concat("Renavam deve conter 11 dígitos\n");

        // ano
        if(automovel.getAno().equals(""))
            mensagem = mensagem.concat("Ano não pode estar vazio\n");
        else {
            try {
                int ano = Integer.decode( automovel.getAno() );
                if(ano < 1900 || ano > 2015) {
                    mensagem = mensagem.concat("Ano deve ser entre 1900 e 2015\n");
                }
            } catch (Exception e) {
                mensagem = mensagem.concat("Ano deve ser um inteiro válido\n");
            }
        }

        // marca
        if(automovel.getMarca().equals(""))
            mensagem = mensagem.concat("Marca não pode estar vazio\n");

        // modelo
        if(automovel.getModelo().equals(""))
            mensagem = mensagem.concat("Modelo não pode estar vazio\n");

        // cor
        if(automovel.getCor().equals(""))
            mensagem = mensagem.concat("Cor não pode estar vazio\n");

        // placa
        if(!automovel.getPlaca().substring(0, 3).matches("[A-Za-z]{3}")
                || !automovel.getPlaca().substring(4, 8).matches("[0-9]{4}"))
            mensagem = mensagem.concat("Placa deve conter 7 caracteres\n");

        // chassi
        if(!automovel.getChassi().matches("[0-9A-Za-z]{17}"))
            mensagem = mensagem.concat("Chassi deve conter 17 caracteres\n");

        // proprietário
        if(automovel.getProprietario() == null)
            mensagem = mensagem.concat("Proprietário não pode estar vazio\n");

        // A nova mensagem dentro do objeto será a mensagem atual
        // com as novas informações
        // Verifica de erro para quando mensagem estiver vazia
        String mensagemAtual = ( automovel.getMessage() == null )? "": automovel.getMessage();
        String mensagemNova = mensagemAtual.concat(mensagem);
        automovel.setMessage(mensagemNova);

        // Caso alguma regra não tenha sido cumprida, há erro e a mensagem não é vazia
        return mensagem.equals("");
    }

    // Objeto1 (da Classe) tem os critérios da busca, caso algum existe
    // Objeto2 (String) especifica o tipo da pesquisa
    public static ArrayList<Automovel>  buscarAutomovel(Automovel automovel, String tipo) {
        // Tratando pesquisa por Renavam
        if(tipo.equals("RENAVAM") && automovel.getRenavam().length() > 0) {
            boolean renavamValido = checaFormatoRenavam(automovel.getRenavam());
            // Se o que foi digitado na pesquisa não for válido, não é necessário fazer a busca
            if(!renavamValido)
                return null;
        }

        ArrayList<Automovel> newList = AutomovelModel.buscarAutomovel(automovel, tipo);

        // Se não houve nenhum erro na pesquisa, e ainda assim, a lista está vazia
        if(!automovel.isError() && newList == null)
        {
            automovel.setError(true);
            automovel.setMessage("Nenhum possível titular foi encontrado.");
            return null;
        }
        else
            return newList;
    }

    private static boolean checaFormatoRenavam(String renavam) {
        return renavam.matches("[0-9]+");
    }

    public static int buscaIDAutomovel(ArrayList<Automovel> automovelList, Automovel automovel) {
        int id = 0;
        for (int i = 0; i < automovelList.size(); i ++)
        {
            Automovel autoVO = (Automovel) automovelList.get(i);
            if(autoVO.getIdAutomovel() == automovel.getIdAutomovel()){
                //System.out.println("autoVO.getIdAutomovel() == automovel.getIdAutomovel()" +
                        //autoVO.getIdAutomovel() + "==" + automovel.getIdAutomovel());
                id = i;
                break;
            }

        }
        //System.out.println("id " + id);
        return id;
    }

}

redpajama_set_name: RedPajamaGithub


text:
HVS Asia Pacific Hospitality Newsletter - Week Ending 27 December 2019 (December 30, 2019)
PAG and Inmark Acquire Grand Hyatt Seoul for KRW560 Billion in Korea
Hong Kong-based investment firm, PAG and Seoul-based Inmark Asset Management has formed a consortium to acquire the iconic 615-key Grand Hyatt Seoul for KRW560 billion (USD480 million). The hotel was sold subject to a long-term management agreement with US-based Hyatt Hotels Corporation and will continue to operate under the Grand Hyatt brand. The property occupies over 73,000 square metres of freehold land in the heart of Hannam-dong, one of the most affluent neighbourhoods in Seoul. Opened in 1978, the property features 12 restaurants and bars, a spa, and 19 meeting spaces. After the renovation completion in May, the hotel saw an addition of 14 rooms, an extensive transformation of all guestrooms and suites, as well as its signature Grand Ballroom. The property also has a long-standing reputation as one of the most luxurious hotels in the city. The acquisition comes after a joint venture between Singapore-based Datapulse Technology and Bizcentre Capital, a unit of Malaysia-based developer, Plenitude Berhad, in April purchased the 224-room Holiday Inn Express Euljiro hotel in Seoul for KRW59 billion (USD50.8 million).
Japan's Haneda Airport to Open Haneda Airport Garden Mixed-use Complex in 2020
Haneda Airport Garden, a new mixed-use complex is set to open in Japan's Haneda Airport in 2020. The project, co-developed by Japan-based real estate company, Sumitomo Realty and Development, and Haneda Airport Development, will comprise 2,400 square metres of events and meeting space, a 2,000-square-metre hot spring facility, a bus terminal offering 15 bus routes connecting Haneda Airport and other parts of Japan, as well as a shopping mall with 60 retail outlets and 30 restaurants. The complex will also feature two airport hotels, the 160- key Villa Fontaine Premier Haneda Airport and 1,557-key Villa Fontaine Grand Haneda Airport. The mixed-use facility will be directly connected to the airport's international terminal and will be approximately one-minute walk from both the Haneda Airport International Terminal Station on The Keikyu Line and the Tokyo Monorail's Haneda Airport International Building Station.
Vietnam and Myanmar Signs Visa-Free Agreement
Starting from January 2020, Myanmar and Vietnam travellers can visit each other's country for 30 days without visas. The agreement is concluded in a bid to make it easier for travellers who want to stay longer in the other country. Currently, Myanmar eased visa requirements for citizens of all members of the Association of Southeast Asian Nations (ASEAN), except Malaysia. Thailand account for most of the tourists from ASEAN countries, followed by Vietnam. The countries visited most often by Myanmar travellers are Thailand, Singapore and Vietnam. There has also been increasing demand for tour packages to Japan, South Korea, India and England.
Philippine Airlines Launched Direct Flights between Manila and Perth
Philippine flag carrier, Philippine Airlines, will launch a new four-times-weekly direct flights between Manila and Perth, starting from March 2020. Presently, passengers must connect in Singapore, Hong Kong or other Australian cities in order to fly between the Philippines capital and the capital of Western Australia ("WA"). The flights will be operating between Manila Ninoy Aquino International Airport and Perth Airport on Mondays, Tuesdays, Thursdays and Fridays. The airline will operate the service on an Airbus A321, which has 168 seats, including 12 fully-flat business class seats in a 2-2 configuration. The new service is expected to inject an additional 70,000 seats into the market. Perth will be Philippine Airlines' fourth destination in Australia, following Sydney, Melbourne and Brisbane, which are all served as direct flights. According to the latest International Visitor Survey, the number of visitors from the Philippines travelling to WA grew by 34.7% in the year ending June 2019.
Royal Air Maroc to Unveil First Direct Route to China
Maroc-based Royal Air Maroc has recently announced that it will launch its first regular direct route between Mohammed V International Airport in Casablanca, Morocco, and Beijing Daxing International Airport in the capital of China. Starting on 16 January 2020, the direct flights will be operational three times a week, serviced by a 302-seat Boeing 787-9 aircraft with 26 business-class seats. The flights to Beijing will depart from Casablanca at 17:00 local time on Mondays, Thursdays and Saturdays, arriving in Beijing at 11:55 local time the next day. The return flights will take off from Beijing at 13:55 on Tuesdays, Fridays and Sundays, arriving in Casablanca at 19:55. The new route will not only facilitate the development of Maroc into a world's leading tourism market but also further enhance the commercial and economic ties between Maroc and China.
Absolute Share Price Performance, as on 27 December 2019
Closing Share Price as at 27 December 2019 20 December 2019 % Change
Elanor Investors Group 2.05 2.12 -3.5%
Event Hospitality & Entertainment Ltd 13.49 13.37 0.9%
General Property Group 5.72 5.82 -1.7%
Mirvac Group 3.21 3.24 -1.0%
Central Plaza Hotel Public Co Ltd 24.60 26.00 -5.4%
The Erawan Group Public Co Ltd 5.65 5.90 -4.2%
Laguna Resorts & Hotel Public Co Ltd 41.00 41.75 -1.8%
Jinling Hotel Corporation Ltd 9.23 9.41 -1.9%
Huatian Hotel Group Co Ltd 2.60 2.63 -1.1%
Guangzhou Dong Fang Hotel Co Ltd 7.40 7.49 -1.2%
Regal Hotels International Holdings Ltd 4.18 4.18 0.0%
The Hong Kong & Shanghai Hotels Ltd 8.50 8.62 -1.4%
Shangri-La Asia Limited 8.33 8.13 2.5%
Chalet Hotels Ltd 340.15 340.55 -0.1%
IHCL (Taj Hotels, Resorts & Palaces) 143.6 142.0 1.1%
Hotel Leela Ventures 5.9 6.0 -1.7%
Lemon Tree Hotels Ltd 64.3 60.2 6.7%
ARA HTrust (US$) 0.87 0.86 1.2%
CDL Hospitality Trusts 1.61 1.62 -0.6%
Eagle HTrust (US$) 0.55 0.56 -1.8%
Red Planet Japan Inc 114 114 0.0%
Mandeep S. Lamba, President, South Asia – HVS Anarock [email protected]
Mei Leng Ho, Senior Vice President – HVS Singapore [email protected]
Florian Kittler, Managing Director – HVS Executive Search Europe & Asia Pacific [email protected]


The Appalachian and Ohio's system map remains unchanged from 1.0. However the new layout has now located the industrial city of Millport across the Ohio river into the Buckeye state. This allowed me to model some Ohio River shoreline after leaving Millport and before jumping across the Ohio on a series of bridges. Several new town locations have shown up given the larger space. And the addition of Bob Sobol's New River and Western connection adds more interchange traffic from the south. The NR&W is a wholly owned subsidiary of the A&O.
The track plan below was a surprise and gracious gift from Jose Alvira from the New York area. If you wish a close up view of the various text items you can do so by the following download. Note that the left hand portion of the new layout is double decked, with the staging yards being below the main yards of Millport, O. Thus the far left portion below lies above the Willow Creek/Taft and Dogtown staging yards. Connection from the lower staging yards into Millport is made at CM Tower at one end and continuous running through to Willow Creek and on to Sobol Springs on the other. The schematic and grade profiles approximate the overall track flow.
As the railroad is being built several changes have and will occur. For instance the coal barge loading at Bayfield have been moved to Glen Forge, and the paper mill at Glen Forge is now at Bayfield. Another passing siding has been added on the Coal Ridge grade encompassing the River Dock loader. Etc. The overall plan remains as depicted below.


text:
© Steffi Henn
Mandy Fredrich
– World management –
In the 2022/23 season, soprano Mandy Fredrich returns to the Teatro del Maggio Musicale Fiorentino in Beethoven's 9th Symphony under the baton of Zubin Mehta. This season Fredrich also debuts at the Staatsoper Berlin as Gutrune (Götterdämmerung) in Dmitri Tcherniakov's new production under the musical direction of Christian Thielemann and makes her house debut in Lyon as Contessa.
In the 2021/22 season, Fredrich appeared at the Teatro del Maggio Musicale Fiorentino in Beethoven's 9th Symphony, Haydn's Die Schöpfung and in Schubert's Messe in Es-Dur under the baton of Zubin Mehta. She made her house debut at The Royal Danish Opera as Marguerite in Gounod's Faust at a very short notice, before returning to Copenhagen as Donna Anna in Mozart's Don Giovanni, conducted by Julia Jones and directed by John Fulljames.
Other important recent engagements have included the Queen of the Night (Die Zauberflöte), Marzelline (Fidelio) and Frau Fluth in a new production of Otto Nicolai's Die lustigen Weiber von Windsor under the baton of Daniel Barenboim at the Berlin State Opera, and her house debuts at the Vienna State Opera and the Royal Opera House Covent Garden as Marguerite/Faust. At La Scala in Milan she had a great success with three operetta gala concerts.
Mandy Fredrich celebrated her international breakthrough at the Salzburg Festival as the Queen of the Night under the baton of Nikolaus Harnoncourt. This was followed by engagements at the Zurich Opera House, La Scala in Milan, the Deutsche Oper Berlin, the Bavarian and Hamburg State Operas, the New National Theatre in Tokyo and the Bregenz Festival, to name but a few. At the Stuttgart State Opera she sang many great roles of the lyric soprano part (Donna Anna, Contessa, Fiordiligi, Marguerite, Micaëla, Antonia, Iphigénie en Aulide).
Mandy Fredrich is also an internationally sought-after artist as a concert singer. She has made guest appearances at the Teatro Verdi in Trieste (Orchesterlieder by Richard Strauss), at the Lugano Festival (Mendelssohn's Lobgesang), with the Dresdner Kreuzchor (Mozart's Requiem, Brahms' Ein Deutsches Requiem) as well as at the Leipzig Gewandhaus at the Maggio Musicale Fiorentino and at La Scala in Milan. She has worked with conductors such as Zubin Mehta, Daniel Barenboim, Christian Thielemann, Julia Jones, Nikolaus Harnoncourt.
Mandy Fredrich comes from Rädigke in Fläming and studied at the UdK in Berlin with Prof. Robert Gambill and at the HMT Leipzig with Prof. Regina Werner-Dietrich as well as with Margreet Honig and Renata Scotto. In 2010 she won the Competizione dell'Opera in Dresden and received the 2nd prize at the Concours Suisse Ernst Haefliger in Gstaad/Bern.
September 2022 – For the most recent biography, please contact Bartosz Jakóbczak
REPERTOIRE OPERA
REPERTOIRE CONCERT


If there has ever been a time to pick up a new shave cream to coincide with coming of summer it is with Elvado's Royal Tahitian Lime Shave Cream. It screams summer. I have been a fan of Elvado's products for some time now and their excellence in both quality and scent selection has never let me down.
Elvado's most recent offering is the Royal Tahitian Lime Shave Cream, which will become available in June, perfect timing for the summer. The only other Lime scented shave cream that I have tried is Taylor of Old Bond Street Lemon and Lime offering, although both lime the two are quite different. The TOBS is more citrusy whereas the fragrance coming from this shave cream is like something from the dessert bar, probably best described as smelling like key lime pie. If the thought of putting a dessert scented cream on your face is unappealing don't let it, it really is a very refreshing way to start your day.
As I mentioned, Elvado's shave creams have always maintained a high consistent standard, regardless of fragrance, and the same has to be said of the Tahitian Lime offering. Lots of cushion, a slick surface, easy to lather (don't be afraid of using some water), and a face that is not irritated.
If you are more of a traditionalist when it comes to shaving products, more 'manly' smells, then maybe Elvado's Tahitian Lime Shave Cream would not be your cup of tea. But if you like to experiment and experience some less traditional scents then you shouldn't hesitate giving this a try, especially while the sun is beating down on us – a perfect summer shave cream. Keep an eye out on Elavdo's web site for availability, or subscribe to our mailing list below to receive an update on this and other items we come across.


text:
John Mayer's Martin 000-ECHF Bellezza Nera
This guitar was seen in the music video for the song 'Half of My Heart' from the 2009 album, 'Battle Studies', and on a home video that John recorded in Japan in 2010. [John Mayer – Half of My Heart (Tokyo Acoustic Version)] Unfortunately, we couldn't find out if the guitar was used on the studio sessions.
The Martin 000-ECHF, nicknamed Bellezza Nera (meaning Black Beauty in Italian), is an Eric Clapton signature guitar introduced in 2004. The guitar features a solid Italian Alpine spruce top with scalloped 5/16″ bracing, East Indian Rosewood back and sides, ivoroid binding, fine herringbone top trim, custom pearl rosette, ebony bridge, and a custom V-shape neck.
If you're curious about the original version used by Eric Clapton, on which this model based on, refer to Eric Clapton's Martin 000-ECHF "Bellezza Nera".
2003 Martin OM-28JM Limited Edition
This guitar was introduced in late 2003 as the first official John Mayer Signature model from Martin. The model was produced as a limited run of 404 instruments, with the #4 and #43 allegedly going to Mayer [source needed]. In the interview with Martin guitar at the 2014 NAMM show, John does mention that he still has the same two OM-28JMs, so that does somewhat confirm the theory that there's more than just […]
Martin DM3MD
This guitar was used by John to record the entirety of the 'Room for Squares' album and during the 2001 tour. The guitar is a limited edition Dave Matthews signature model from Martin. According to the official specs, it features a 14 fret dreadnought body with solid spruce top and solid East Indian rosewood back and sides, solid mahogany neck with ebony fretboard, and grained ivoroid body binding.
National Style-O Resonator 12-string
John used this guitar on the rare occasions when playing the song 'Walt Grace's Submarine Test, January 1967' live (something he initially avoided doing but does more often in recent years). This National seems to be a custom made guitar since it looks to be fairly new, and features a 12-string neck – which seems to be very rare on these guitars. Next to that, the guitar is also equipped with a sound […]
Martin 000-14 Cutaway Custom
This Martin was mostly used just for the 2001 Room for Squares tour, it being one of only two Martins that John was seen playing around that time (the DM3MD being the other one). It was likely picked up just due to fact that it was a cutaway guitar, and allowed John to reach the higher frets more easily while playing live. Unfortunately, trying to figure out the exact model of […]
John was seen playing this guitar on occasion in early 2003, most notably at the 45th Annual Grammy Awards show on February 23rd. The guitar is one of Martin's premium models with $5,000+ price tag, featuring Sitka spruce top, East Indian rosewood back and sides, Abalone rosette and the top inlay, and grained ivoroid body and headstock binding.
Martin 00-45SC
This is the second guitar that John designed working with together Martin, introduced initially at NAMM 2012. According to John's own words, the idea behind the guitar was to create a smaller and a more portable guitar. The guitar features a 00-style body with an Adirondack spruce top and Cocobolo back and sides (notice that all the prior Martins that John played had East Indian rosewood back and sides), and a select hardwood neck with ebony […]
Martin D-45 JM
Martin released this guitar in early 2018, as the first dreadnought size John Mayer signature model. It was produced in a limited run of only 45 guitars, and it featured Engelmann Spruce top with Guatemalan Rosewood back and sides, and Abalone top and rosette inlays. At the time of the release, the Martin D-45 JM sold for $14,999.00.
Martin OM28-V
By 2002 Mayer seems to have decided to start moving from a dreadnought sized guitars to OM/000 sized ones. For the first guitar, the choice fell on an OM28-V model, which is part of Martin's Vintage Series line. This guitar was mostly seen just during the 2002 tour, and by 2003 it was replaced by Mayer's own signature model. Martin OM28-V features a solid Sitka spruce top with solid East Indian rosewood back and […]
Washburn Acoustic
In his Periscope stream on August 20th, 2015 [John Mayer on Periscope – Gear Talk] John mentioned that his first guitar was a Washburn acoustic and that the guitar is still with his family. Unfortunately, this is the only piece of info available on this guitar, so if you happen to come across some old photos of John with it, or an interview where he happens to mention it, please […]
Godin Multiac
John was seen using this guitar only on one video clip dating back to March 9, 2000 [John Mayer – Live from the Gothic Theatre, Denver, March 9, 2000]. The origin of the guitar and the time frame during which John used it are currently unknown.


The Republican National Convention may be confined to the walls of Quicken Loans Arena (The Q) this July, but the Cleveland 2016 Host Committee, responsible for organizing, hosting and funding the 2016 Republican National Convention in Cleveland, is getting out into the community to explain how engaging with and supporting the Convention is more about taking pride in hosting 50,000 visitors in our city. To do this, the 2016 #ThisisCLE Mobile Tour is traveling throughout the city.
Since its debut in 2014, Destination Cleveland's #ThisisCLE Mobile Tour has attracted thousands of residents to celebrate their city pride. Now, the convention and visitors bureau for Greater Cleveland has partnered with the Cleveland 2016 Host Committee to take the 2016 #ThisisCLE Mobile Tour on the road. Launched at the Cleveland Cavalier's first Round 1 postseason game against the Detroit Pistons at The Q on Sunday, April 17, the mobile tour's Cleveland-themed truck with patriotic wrap highlights the Host Committee's role in the Convention preparation as being about Cleveland pride, not politics. The iconic larger-than-life letters that spell out "#ThisisCLE" also travel with the tour.
The goal of the tour is to educate Northeast Ohio residents about the impact the 2016 Republican National Convention will have on Cleveland and its future. The Host Committee is the non-political organization responsible for organizing, hosting and funding the 2016 Republican National Convention.
May 15: Rite Aid Cleveland Marathon (at the finish line, 7am-1pm).
May 21: Cleveland Asian Festival (time still TBD).
June 4: Legacy Village Art Show, Beachwood (noon-4pm).
June 26: Crocker Rocks event at Crocker Park, Westlake (3pm-7pm).
Locals can follow the Mobile Tour on Twitter at @HappyinCLE and @2016CLE.