ORIGINAL RESEARCH article
Integration of Metagenomic and Stable Carbon Isotope Evidence Reveals the Extent and Mechanisms of Carbon Dioxide Fixation in High-Temperature Microbial Communities
- 1Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT, USA
- 2Thermal Biology Institute, Montana State University, Bozeman, MT, USA
- 3Pacific Northwest National Laboratories, Richland, WA, USA
Although the biological fixation of CO2 by chemolithoautotrophs provides a diverse suite of organic compounds utilized by chemoorganoheterotrophs as a carbon and energy source, the relative amounts of autotrophic C in chemotrophic microbial communities are not well-established. The extent and mechanisms of CO2 fixation were evaluated across a comprehensive set of high-temperature, chemotrophic microbial communities in Yellowstone National Park by combining metagenomic and stable 13C isotope analyses. Fifteen geothermal sites representing three distinct habitat types (iron-oxide mats, anoxic sulfur sediments, and filamentous “streamer” communities) were investigated. Genes of the 3-hydroxypropionate/4-hydroxybutyrate, dicarboxylate/4-hydroxybutyrate, and reverse tricarboxylic acid CO2 fixation pathways were identified in assembled genome sequence corresponding to the predominant Crenarchaeota and Aquificales observed across this habitat range. Stable 13C analyses of dissolved inorganic and organic C (DIC, DOC), and possible landscape C sources were used to interpret the 13C content of microbial community samples. Isotope mixing models showed that the minimum fractions of autotrophic C in microbial biomass were >50% in the majority of communities analyzed. The significance of CO2 as a C source in these communities provides a foundation for understanding community assembly and succession, and metabolic linkages among early-branching thermophilic autotrophs and heterotrophs.
Despite the significance of microorganisms in nearly all major element cycles, the contribution of microbial inorganic carbon (C) fixation to global C cycling is not yet well-resolved (Shively et al., 2001; Reinthaler et al., 2010). Resolution of the C cycle is important across multiple scales, ranging from specific microbial communities to regional environments, and ultimately, to the global Earth system. On an energetic basis, the reduction of CO2 to biomass C is extremely costly and organisms obtain this energy either from photons (i.e., phototrophy) or the oxidation of reduced chemical species (i.e., chemotrophy). The plethora of inorganic electron donors that are available to drive chemolithoautotrophy include reduced forms of hydrogen, sulfur, nitrogen, arsenic, carbon, iron, and manganese. The fixation of CO2 occurs via one of six currently known biochemical pathways in Archaea, Bacteria, and/or Eukarya. Therefore, resolution of C cycling at the microbial community scale requires the integration of metabolic and molecular data (e.g., metagenome sequence) with geochemical studies, such as stable carbon isotope (13C) analysis or isotope probing. Stable isotope probing (SIP) using 13CO2 and subsequent analysis of heavy DNA allows for the identification of organisms responsible for the fixation of CO2 in microbial communities; however, SIP is technically challenging in situ across a large number of different environments. Moreover, other measurements are required to gain appreciation for the relative extent of autotrophy vs. heterotrophy in mixed microbial communities. Direct analysis of stable carbon isotopes (i.e., 13C) has been employed to dissect the trophic structure of numerous different ecosystems, and can provide primary evidence for the sources of C to specific biota (Peterson and Fry, 1987).
Currently established pathways responsible for the fixation of CO2 in chemotrophic organisms that are known inhabitants of iron-oxide, sulfur-sediment, and filamentous “streamer” communities in Yellowstone National Park (YNP, Wyoming, USA) include the reductive tricarboxylic acid cycle, reductive acetyl-CoA pathway, 3-hydroxypropionate/4-hydroxybutyrate cycle, and the dicarboxylate/4-hydroxybutyrate cycle (Berg et al., 2010; Hügler and Sievert, 2011). The reverse tricarboxylic acid (r-TCA) cycle has been described in numerous lineages of Bacteria, including the hyperthermophilic Aquificales, which are known to inhabit marine hydrothermal vents and geothermal environments (Reysenbach et al., 2000, 2005; Huber and Eder, 2006). The reductive acetyl-CoA (Wood-Ljungdahl) pathway has been described in acetogenic bacteria and methanogenic archaea, as well as sulfate-reducing bacteria (Hügler and Sievert, 2011), which are found in hypoxic environments including sediments, soils, sludge, and intestinal tracts (Breznak and Kane, 1990; Whitman et al., 2006; Drake et al., 2008). The 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle has been identified in several major lineages of Archaea, including the Crenarchaeota (order Sulfolobales) and marine Thaumarchaeota (Berg et al., 2007; Walker et al., 2010; Könneke et al., 2014). The dicarboxylate/4-hydroxybutyrate (DC/4-HB) cycle utilizes a PEP carboxylase and has been identified in the Thermoproteales and Desulfurococcales (phylum Crenarchaeota; Huber et al., 2008; Ramos-Vera et al., 2009); the regenerative half of this cycle (i.e., succinyl-CoA to acetyl-CoA) is identical to the 3-HP/4-HB cycle.
Energy-rich hydrothermal and geothermal environments contain deeply-branching thermophiles basal to the tree of life and are thought to be analogous to environments potentially common on early Earth (Nisbet and Sleep, 2001). Numerous high-temperature geothermal environments in Yellowstone National Park (YNP, WY, USA) have been characterized (e.g., Inskeep et al., 2013a) and provide opportunities for examining non-phototrophic CO2 fixation across a range of habitat types. Prior efforts to determine the importance of CO2 fixation in high-temperature microbial communities have included stable isotope measurements (Estep, 1984; Jahnke et al., 2001; Havig et al., 2011), 14CO2 incorporation assays (Boyd et al., 2009), and genome-enabled analysis of microbial communities and important isolates (Kozubal et al., 2008; Inskeep et al., 2010, 2013b; Takacs-Vesbach et al., 2013). However, there have been no systematic studies reporting on the 13C content of microbial community biomass present in non-phototrophic thermal environments. Consistent δ13C (‰) values of dissolved inorganic and organic C (i.e., DIC and DOC) have averaged approximately −3 and −23%, respectively, across different geothermal springs in YNP (Craig, 1953a,b; Bergfeld et al., 2014). We recently utilized the large difference in 13C content between inorganic and organic C pools to evaluate the extent of CO2 fixation in acidic Fe(III)-oxide microbial mats (Jennings et al., 2014), and showed that CO2 fixation contributed no less than ~40% of the microbial C in biomineralized Fe-oxide mat communities.
Here, we expand this approach to integrate random metagenome sequence analysis to identify the relative abundance of candidate autotrophs, and their respective CO2 fixation pathways, then utilize this information to evaluate the extent of CO2 fixation using stable 13C analysis of biomass C, dissolved inorganic C and organic C content of 15 different high-temperature microbial communities in YNP where phototrophic organisms are not important community members. The primary objectives of this study were to (i) determine the stable 13C isotope contents of dissolved inorganic and organic C, landscape organic C, and microbial mat or sediment C from major types of geothermal environments in YNP, (ii) identify the relative abundance of different chemotrophic microbial populations capable of CO2 fixation across sites, as well as the respective pathways potentially responsible for this metabolism, and (iii) evaluate the extent of microbial CO2 fixation in chemotrophic communities using isotope mixing models constructed with information from metagenome sequence data of these same sites. Results showed that CO2 fixation is an important metabolic process in nearly all high-temperature (T ~ 70–85°C) chemotrophic systems studied, which included sulfur-sediments, Fe(III)-oxide mats, and filamentous “streamer” communities ranging in pH from 2.5 to 9.
Materials and Methods
The geothermal samples (Table 1, Table S1) obtained in the current study encompass a wide range of chemotrophic environments that capture major phylogenetic groups known to inhabit geothermal systems of YNP (Inskeep et al., 2010, 2013a,b; Kozubal et al., 2012; Takacs-Vesbach et al., 2013; Beam et al., 2016a,b; Jay et al., 2016). These sites have been the subject of extensive prior characterization and represent three distinct habitat types: hypoxic sulfur sediments, Fe(III)-oxide microbial mats, and filamentous “streamer” communities. The pH and temperature of these geothermal environments range from 2.0 to 9.4, and 68–87°C, respectively (Table 1).
Table 1. Key geochemical parameters, and 13C stable isotope content (δ, ‰) of dissolved inorganic C, dissolved organic C (DIC, DOC) and microbial mat C1 determined across 15 different high-temperature springs in Yellowstone National Park (WY, USA).
Carbon Stable Isotope Analysis
Water samples were collected on site, filtered (0.2 μm) into sterile Falcon tubes, and analyzed for isotope content (13C) and total concentration of dissolved inorganic and dissolved organic C at the Colorado Plateau Stable Isotope facility (Flagstaff, AZ). Solid phase sediment, mats, and/or streamer samples were obtained using a sterile spatula, placed into sterile 50 mL Falcon tubes, and frozen until analysis. Samples used to evaluate the isotopic composition of “landscape” carbon were obtained using the same protocols from surrounding soils, vegetation, animal dung, and algal mats. Numerous sites were sampled multiple times over 3 years (details for each geothermal location are provided in Table S1).
Stable carbon isotope ratios (δ13C) of mat biomass and landscape samples were measured using a Thermo-Finnegan (Bremen, Germany) Delta V Plus isotope ratio mass spectrometer coupled to a Costech Analytical Technologies (Valencia, CA, USA) 4010 Elemental Analyzer and a Zero-Blank autosampler. Approximately 8–15 mg of lyophilized sediment, mat, streamer, or landscape sample was placed into tin capsules (Costech Analytical Technologies) and analyzed. Samples containing calcium carbonate (i.e., Conch and Narrow Gauge) were pre-treated overnight in 1 M HCl to remove carbonate minerals. Data were corrected using the point-slope method (Coplen et al., 2006) to in-house Pacific Northwest National Laboratory (PNNL) glutamic acid standards, which were calibrated to U.S. Geological Survey (USGS) Standard 40 (δ13C = −26.39%) and USGS Standard 41 (δ13C = +37.63%) glutamic acid isotopic standards. Carbon stable isotope content is measured as a ratio, R (13C/12C), then reported as a delta (δ) value, where δ equals (RSA/RStd − 1) × 1000 and RSA and RStd are the isotope ratios of the sample and the internationally recognized standard, PeeDee Belemnite (PDB).
Random Metagenome Analysis of Predominant Phylotypes
Assembled genome sequences of numerous thermophilic microorganisms from YNP geothermal environments (primarily genus level) were constructed primarily from Sanger and/or pyro-sequencing (Kozubal et al., 2012, 2013; Inskeep et al., 2013a,b; Takacs-Vesbach et al., 2013; Jay et al., 2016). These metagenome sequence assemblies were annotated using protocols established at the DOE-JGI in the IMG/Expert Review system (Markowitz et al., 2012), and have been deposited with the Department of Energy Joint Genome Institute (DOE-JGI) Integrated Microbial Genomes/Metagenome site (IMG/M) under corresponding Genome ID values (see Table S2 for full list of phylotypes). These sequence assemblies were used as high-quality reference sequences to bin random Illumina metagenome sequences from many of the same sites, perform relative abundance calculations, and to assess the potential for CO2 fixation based on genes identified in known biochemical pathways. Results for the reverse TCA, 3-HP/4-HB and/or DC/4-HB cycles (Beh et al., 1993; Hügler et al., 2005; Berg et al., 2007; Ramos-Vera et al., 2009) are summarized in Supplemental Information (Tables S3, S4). Significant evidence of the Calvin and 3-HP cycles was not observed in the organisms and habitats studied, though forms of RubisCO can occasionally be identified in metagenomes of high-temperature systems. The lack of evidence for the Calvin and 3-HP cycles is consistent with the absence of phototrophic microorganisms (e.g., Cyanobacteria and Chloroflexi). Microbial populations potentially utilizing the reductive acetyl-CoA pathway (i.e., acetogens, methanogens, and sulfate-reducing bacteria) were not dominant members of these communities, and genes coding for marker proteins in these pathways were not observed consistently in the reference sequence assemblies obtained from these sites. Consequently, the Calvin and 3-HP cycles, and the reductive acetyl-CoA pathway were not highly-represented in the habitats studied and were not considered as possible mechanisms contributing to CO2 fixation.
Relative Abundance of Different Inorganic C Fixing Microorganisms
Additional microbial mat samples from the same sites were transported on dry ice, and stored at −80°C prior to DNA extraction, and subjected to random Illumina metagenome sequencing at the DOE-Joint Genome Institute (Community Sequencing Program 701) (Table S5). These samples were also subjected to 16S rRNA gene amplicon sequencing using Illumina barcoded samples (iTags). Random metagenome sequence reads from Illumina sequencing were compared to de novo genome assemblies (or isolate genomes) generated previously using Sanger and/or 454 pyrosequencing (Inskeep et al., 2013a,b; Takacs-Vesbach et al., 2013). Individual sequence reads were binned (at 90% nucleotide identity) to respective phylotype genome assemblies and the relative abundance (RA) of individual populations (genera level or higher) was estimated as the percent of sequence reads binned compared to the total sequences available. Although not all phylotypes across all sites have been curated, with the exception of Washburn Spring, ~70–90 percent of individual random sequences were assigned to well-characterized phylotypes at 90% nucleotide identity (Table S6). Results from deep 16S rRNA gene sequencing (Illumina iTags) were used for determining relative population abundances in two sites. In the these cases, operational taxonomic units (OTU) were established at 97% nucleotide identity for short fragment iTag sequences, and then identified at >90% nucleotide identity by comparison to an internal reference set of long-fragment (>1200 nt) 16S rRNA gene sequences from these same geothermal features in YNP. All long-fragment reference 16S rRNA gene sequences are available on NCBI.
13C Mixing Models
The 13C stable isotope contents of microbial mat samples were predicted using site-specific mixing models, formulated on δ13C values of dissolved inorganic C (DIC), dissolved organic C (DOC), and landscape organic C (OC), as well as the weighted fractionation factor based on the relative abundance of CO2-fixing microorganisms in the community. The measured δ13C of sulfur sediments, Fe(III)-oxide mats, and filamentous “streamer” communities may contain C from microbial biomass of DIC-origin, microbial biomass of OC-origin, and non-thermophilic biomass of landscape organic C. Given the range of observed δ13C-DIC values (−5.4 to 5.4%) and the statistically similar δ13C content of DOC and exogenous organic C (mean = −22.8%, σ = 3.0), 13C mixing models were developed for each system based on the measured δ13C-DIC, and the mean δ13C of dissolved organic carbon (DOC) and landscape C for all systems. The stable carbon isotope contents (13C) of all samples were predicted as a function of the ratio of microbial biomass of DIC-origin relative to total microbial biomass (fMicrobial-DIC, where fMicrobial-DIC + fMicrobial-DOC/OC = 1), and the ratio of biomass C relative to total mat C (fMicrobial-C, where fMicrobial-C + fNon-microbial-C = 1) using the following relationships:
Weighted fractionation factors used in the mixing models were based on the relative abundance of each CO2-fixing microorganism and the predicted autotrophic fractionation factor (ε-value) for each phylotype (Table 2); fractionation factors were estimated based on carboxylase sequence similarity with cultured relatives (Jahnke et al., 2001; House et al., 2003; Jennings et al., 2014). For modeling purposes, the heterotrophic fractionation of 13C-OC (i.e., DOC or landscape OC with an average 13C content of ~ −23%) into microbial biomass was assumed to be 0% (Boschker and Middelburg, 2002).
Table 2. Relative abundance of primary autotrophs distributed across different geothermal habitats in Yellowstone National Park and predicted fractionation factors (ε) for the three major carbon dioxide fixation pathways observed in these populations.
13C Content of DIC, DOC, and Landscape OC
The 13C contents of DIC and DOC measured across 15 geothermal systems averaged −2.7% (± 2.8) and −21.7% (± 2.3), respectively (Figure 1). The 13C contents of each C “pool” were fairly well-constrained across geochemically diverse sites with a few exceptions. The larger 13C-DIC value at Crater Hills (5.4%) appeared to be the primary outlier relative to other diverse sites including Mammoth Hot Springs (MHS) where high DIC concentrations (up to 15–20 mM) reflect the dissolution of Madison limestone during ascent of hydrothermal fluids (Fouke, 2011). Most of the δ13C-DOC values from geothermal sites were close to the average δ13C value obtained for landscape organic C (−23.7%), although a few springs exhibited slightly heavier δ13C values near −17 to −18% (Table 1).
Figure 1. Stable carbon isotope contents (δ13C, %) of dissolved organic C (DOC) and dissolved inorganic C (DIC) pools plotted as a function of the concentration of C in 15 geochemically diverse high-temperature environments of Yellowstone National Park, WY, USA.
The majority of geothermal source waters and outflow channels sampled in this study exhibited DIC concentrations that were ~10-fold higher than DOC (Figure 1, Table 1). Most of the observed DOC levels were near the analytical detection limit (~20–40 μM C), which has also made it difficult to obtain compositional data of DOC in these systems. The high concentrations of DIC relative to DOC in these geothermal springs suggest that CO2 fixation may be an important source of biomass C in many high-temperature communities, and may provide reduced C compounds that are utilized by heterotrophic populations.
The 13C isotope content of potential landscape C sources was determined for plant needles, leaves, algal and cyanobacterial communities, animal dung (e.g., bison and wolf) and soils collected from areas near and/or upslope of nearly all geothermal springs sampled. The average δ13C values of landscape samples was −23.7% (n = 29) and ranged from −16 to −28.6% (Table S1), with the exception of Zygogonium spp. algal mat samples (i.e., δ13C = −10.7%), which were not found within the immediate upstream flow path for mats sampled in this study. The average 13C isotope content of leaf, soil, and dung samples (δ13C ~ −24%) reflects the importance of C3 photosynthesis as the primary C source in landscape samples (Ehleringer and Monson, 1993). Landscape OC may contribute to the total C present in geothermal samples either as C utilized to produce microbial biomass and/or as direct landscape inputs.
13C Content of Chemotrophic Microbial Communities
The 13C contents of all microbial community samples were between the average δ13C values for DIC and OC (DOC or landscape OC; Table 1). Nearly all microbial samples exhibited 13C values significantly higher (less negative) than 13C-DOC or 13C-OC from landscape sources, which provides direct evidence for the presence of DIC-derived microbial biomass. Several sulfur sediment communities (i.e., Monarch Geyser, Joseph's Coat Hot Springs [JC3_A, JC2_E]) exhibited 13C values ranging from ~−6 to −16%, and all Fe(III)-oxide sites exhibited 13C values ranging from ~−8 to −16%, which indicate significant fractions of DIC-derived biomass in these microbial communities. The 13C content of filamentous “streamer” samples (containing different members of the Aquificales) showed that these communities also exhibit large, although variable extents of CO2 fixation (Table 1). A more accurate determination of the amount of DIC-derived microbial C present in these communities is possible after considering the appropriate fractionation factors (below) for incorporation of 13C-DIC into microbial biomass, which is dependent on the primary microorganisms present and their respective pathways for CO2 fixation.
Relative Abundance of CO2-Fixing Microorganisms
Bacteria and/or archaea with evidence for known CO2 fixation pathways were identified in the microbial communities studied using random metagenome sequencing (Table 2). The relative amount(s) of autotrophic populations varied considerably across the habitat types evaluated, and ranged from as high as 94% at MHS (a streamer community dominated by Sulfurihydrogenibium spp.), to as low as ~6% in Washburn Spring (sediment community), which contains a diverse mixture of archaeal and bacterial heterotrophs (Inskeep et al., 2013b). Microbial community members not containing established CO2 fixation pathways were lumped into heterotrophic populations (Table S6) for purposes of interpreting 13C stable isotope data; the phylogeny and metabolic attributes of many of these populations are elucidated in more detail in other studies (Inskeep et al., 2013b; Beam et al., 2014, 2016a,b; Jay et al., 2014, 2015, 2016; Colman et al., 2016).
The most common phylotypes that contained CO2 fixation pathways across these habitats included different members of the Sulfolobales (pH < 4), the Thermoproteales (pH 3–9), and the Aquificales (pH 3–9; Table 2). Metallosphaera yellowstonensis and several Sulfolobus-like organisms present in low pH Fe(III)-oxide microbial mats and sulfur-sediments contained key genes of the 3-HP/4-HB pathway, and prior work has shown that M. yellowstonensis str. MK1 is a facultative autotroph in these habitats (Kozubal et al., 2008; Jennings et al., 2014). Caldivirga, Thermoproteus, and Pyrobaculum spp. were the predominant phylotypes with genomic evidence for CO2 fixation in higher pH (pH ~ 5–9) sulfur-sediment systems (Table 2) via the DC/4-HB pathway (Ramos-Vera et al., 2009; Jay et al., 2016). Pathway completeness of the DC/4-HB cycle in the Thermoproteales T1 and T2 phylotypes was difficult to assess based on lower coverage Sanger sequencing (Table S3); however, analysis of Illumina metagenomes from these same sites (Table S4) provided support for complete DC/4-HB CO2 fixation pathways in the Thermoproteales T2 (Pyrobaculum/Thermoproteus) phylotypes. This is consistent with genomic and/or physiologic data of cultured members of these genera (Schäfer et al., 1986; Ramos-Vera et al., 2009; Jay et al., 2015).
Members of the Aquificales were identified in acidic sulfur streamers and Fe(III)-oxide mats (i.e., Hydrogenobaculum spp.), circumneutral sulfidic streamers (Sulfurihydrogenibium spp.), and slightly-alkaline (pH ~ 8–9) streamer communities (Thermocrinis spp.; Table 2). All of the Aquificales phylotypes contained nearly complete pathways necessary for the fixation of CO2 via the reverse TCA cycle, although slight variations exist regarding the mechanism of citrate cleavage among the Aquificales lineages found in YNP (Takacs-Vesbach et al., 2013; Table S3).
Fractionation factors (ε) for the incorporation of CO2 into Metallosphaera biomass and other close Sulfolobales relatives via the 3-HP/4-HB pathway range from 0.2 to 3.6% (House et al., 2003; Jennings et al., 2014). For the DC/4-HB pathway, reported ε-values range from 2.0 to 2.9% in Thermoproteales isolates (House et al., 2003). Fractionation factors for Aquificales isolates range from 3.3 to 5.5%, based on investigation of Hydrogenobaculum and Thermocrinis spp. (Jahnke et al., 2001; House et al., 2003). Consequently, fractionation factors for the primary CO2 fixation pathways identified in the dominant autotrophic populations present in these communities range from ~0.2 to 5.5%.
Extent of Autotrophy
The extent of autotrophy cannot be ascertained directly from the relative abundance of populations with autotrophic potential (i.e., random metagenome data), in part because most of these organisms are not obligate CO2-fixers. These microorganisms are facultative autotrophs and may obtain a fraction of their C from organic carbon sources. Moreover, the presence of landscape OC in a microbial mat sample will result in less 13C, and must be accounted for as a possible contribution to sample 13C values. Consequently, the 13C values of mat, streamer or sediment samples reflect contributions from DIC-derived and OC-derived microbial C, as well as landscape OC (Equation 1); sample 13C values were predicted using mixing models with appropriate fractionation factors for incorporation of DIC or OC (i.e., ε = 0%).
Weighted average fractionation factors (ε) based on the relative abundances of autotrophic populations (Table 2) were used as input values for predicting the sample 13C contents as a function of DIC-derived microbial C relative to total microbial C, and the ratio of microbial C to total mat C (Equation 1). Although the amount of microbial C relative to total mat C is difficult to determine accurately, the measured 13C contents constrain estimates of this ratio as a function of the fraction of DIC-derived microbial C to total microbial C (Figures 2–4). Due to the small range of fractionation factors for conversion of 13C-DIC into microbial biomass compared to the large differences in δ13C-DIC and δ13C-OC, the isotope mixing models were relatively insensitive to the weighted average fractionation factors obtained from the relative abundance of specific autotrophs.
Figure 2. Stable 13C isotope mixing models of sulfur-sediment microbial communities. The fraction of microbial C of DIC origin relative to total microbial C (fMicrobial-DIC) (x-axis) and the fraction of microbial C to total sample C (fMicrobial-C) (y-axis) were used to model (Equation 1) the distribution of possible sample 13C contents (gray scale). Dotted lines indicate possible combinations of fMicrobial-DIC and fMicrobial-C that are consistent with the observed 13C values (‰) of microbial samples (boxed).
With the exception of high-OC sediments in Crater Hills and Washburn Spring (Figure 2), sulfur-sediment communities exhibited significant incorporation of DIC across a wide pH range from 2.0 to 6.2 (Figure 2). For example, the minimum fractions of DIC-microbial C in sulfur-sediments from Joseph's Coat (JC2_E and JC3_A) and Monarch Geyser were >50%. The primary autotrophs in low pH (pH < 3.0) sulfur systems include members of the Sulfolobales and Aquificales (Hydrogenobaculum spp.), and members of the Thermoproteales (i.e., Thermoproteus and Pyrobaculum-like organisms) across a pH range from ~4 to 9 (Table 2). Integration of 13C data and metagenome analysis show that these populations are responsible for the fixation of inorganic C in situ.
The lower fraction of DIC-microbial C relative to total microbial C (x-axis, Figure 2) observed in Crater Hills and Washburn Springs suggests contributions from heterotrophy (incorporation of OC) and/or landscape OC in these samples. Crater Hills is inhabited primarily by Sulfolobales populations, which are capable of CO2 fixation via the 3-HP/4-HB pathway (Table 2), yet the minimum fraction of DIC-microbial C to total microbial C is quite low (< ~20%). Thus, the Sulfolobales populations (2 predominant phylotypes) at Crater Hills may utilize significant amounts of OC, and/or the sample contains a high fraction of non-microbial C from landscape sources. For example, the extent of autotrophy at Crater Hills could exceed 50% of the microbial C if landscape OC were to have contributed >80% of the total sample C (Figure 2, top left). In contrast, Washburn Spring sediments (pH 6.2) contained a much lower relative abundance of autotrophic microorganisms (Table 2). The low 13C contents may reflect high fractions of heterotrophic consumption of OC and/or high fractions of non-microbial C from landscape sources. The Washburn Spring and Crater Hills sites are both located in landscape depressions and receive significant amounts of vegetative debris and surface runoff, which contributes to the large fraction of 13C-OC in these sediments.
The minimum fraction of DIC-microbial C in acidic Fe(III)-oxide mats ranged from near 40–85% (Figure 3), and was consistent across several different types of Fe(III)-oxide systems (Kozubal et al., 2012). Moderately acidic (pH ~ 3.0–3.5), poorly-crystalline, high-arsenic Fe(III)-oxide mats in Norris Geyser Basin (OSP_B, BE_D) were comprised of 40–60% DIC-microbial C. An additional Fe(III)-oxide mat containing less arsenic (Kozubal et al., 2012) also showed significant fractions of DIC-microbial C (ECH_B). Moreover, an extremely mineralized Fe(III)-oxide mat (Kozubal et al., 2012) with crystalline goethite and hematite (JC2_B, pH 2.2) contained a large fraction of organisms capable of CO2 fixation (primarily Sulfolobales), and a minimum of 85% of the microbial C was predicted to be of DIC-origin (Figure 3). Consequently, definitive evidence for significant extents of CO2 fixation was obtained for numerous Fe(III)-oxide communities, and in all cases, metagenome sequence analysis confirmed the importance of Hydrogenobaculum spp. (reverse TCA cycle) and members of the Sulfolobales (e.g., M. yellowstonensis and Stygiolobus-like spp.; 3-HP/4-HB cycle) as the primary autotrophic organisms in these systems, despite differences in pH (~2–4) and Fe(III)-oxide mineralogy (Inskeep et al., 2004; Kozubal et al., 2012).
Figure 3. Stable 13C isotope mixing models of acidic Fe(III)-oxide microbial communities. The fraction of microbial C of DIC origin relative to total microbial C (fMicrobial-DIC) (x-axis) and the fraction of microbial C to total sample C (fMicrobial-C) (y-axis) were used to model (Equation 1) the distribution of possible sample 13C contents (gray scale). Dotted lines indicate possible combinations of fMicrobial-DIC and fMicrobial-C that are consistent with the observed 13C values (‰) of microbial samples (boxed).
Filamentous “streamer” communities were sampled across a wide range of geochemical conditions (pH 3.0–9.4; high H2S vs. high O2), and in all cases, a significant fraction of DIC-microbial C was observed (Figure 4). Different streamer communities included the low-pH systems dominated by Hydrogenobaculum spp. (Dragon Spring), the circumneutral (pH 6.3) springs inhabited by Sulfurihydrogenibium spp. (Mammoth Hot Springs) and the higher pH (8–9) systems (Octopus and Conch Springs) that contain significant fractions of Thermocrinis spp. (Table 2). The fixation of inorganic C by these autotrophs supports a diverse group of different heterotrophic organisms (Table S6) distributed across this habitat range (Takacs-Vesbach et al., 2013; Colman et al., 2016), and demonstrates the importance of the Aquificales and the r-TCA cycle (Table 2, Table S3) in establishing high-temperature filamentous “streamer” communities.
Figure 4. Stable 13C isotope mixing models of thermophilic filamentous “streamer” communities. The fraction of microbial C of DIC origin relative to total microbial C (fMicrobial-DIC) (x-axis) and the fraction of microbial C to total sample C (fMicrobial-C) (y-axis) were used to model (Equation 1) the distribution of possible sample 13C contents (gray scale). Dotted lines indicate possible combinations of fMicrobial-DIC and fMicrobial-C that are consistent with the observed 13C values (‰) of microbial samples (boxed).
The fixation of CO2 provides reduced biomass C to a wide range of heterotrophic assemblages (Table S6) that comprise each of the microbial communities studied. The primary thermophilic bacteria and archaea with known pathways for CO2 fixation, and which were present in sites with significant fractions of DIC-derived microbial C included representatives of the Aquificales (three different genera), as well as the Sulfolobales and Thermoproteales (phylum Crenarchaeota). These chemolithoautotrophs obtain energy for CO2 fixation from the oxidation of reduced species including H2, H2S, S0, As(III), and Fe(II), and are distributed in large part based on their ability to utilize specific electron donor-acceptor combinations. For example, the respiration of autotrophic Sulfolobales and Aquificales populations is coupled with the reduction of O2 (e.g., Inskeep et al., 2013b; Takacs-Vesbach et al., 2013). However, several sub-oxic autotrophic members of the Thermoproteales likely reduce elemental S and/or arsenate (Jay et al., 2015, 2016).
Metagenome and stable isotope analyses were used to show that CO2 fixation occurs across extremely diverse geochemical conditions, including three predominant chemotrophic habitat types in YNP. Inorganic C fixation thus supports both autotrophic and heterotrophic populations in high-temperature communities. The autotroph-heterotroph associations implicated from these 13C stable-isotope measurements, combined with molecular data on community composition and function reveal that three major CO2 fixation pathways are responsible for supporting a diverse array of different heterotrophic populations that are adapted to different geochemical conditions (e.g., pH, oxygen, sulfur). The importance of CO2 fixation in thermal systems also suggests that trophic structure and autotroph-heterotroph interactions are consistent themes in microbial communities often thought to be analogs of environments supporting early life on Earth (Nisbet and Sleep, 2001).
Stable 13C isotope mixing models were used to constrain possible solutions to the observed 13C content of Fe(III)-oxide, sulfur-sediment and filamentous “streamer” samples. Results demonstrated that, at a minimum, 40–50% of the total microbial C in many systems analyzed was derived from the fixation of DIC (x-axis, Figures 2–4). Further resolution of the fraction of non-microbial C (i.e., landscape OC; y-axis, Figures 2–4) would assist in clarifying the extent of autotrophy, and this would be most beneficial in sediment environments of geothermal springs located in landscape depressions, which contain large fractions of OC-derived microbial biomass and/or significant inputs of OC from landscape sources. Geothermal sites in concave topography (e.g., Crater Hills, Washburn Springs) receive significant inputs of landscape OC through erosion, litter-fall and/or storm-water relative to convex systems (e.g., MHS, Conch Spring). Consequently, as it relates to the input of landscape carbon, topographic position is likely an important selective force in the establishment, assembly, and resulting composition of geothermal microbial communities.
Chemoorganoheterotrophs in these high-temperature communities obtain a fraction of biomass C from autotrophic community members (i.e., DIC-derived microbial C), in addition to DOC and/or landscape OC. The similar 13C contents of landscape OC and geothermal DOC preclude the separation of these possible OC sources to microbial community members. Although little is known regarding the composition and availability of DOC in geothermal habitats (and should be addressed in future studies), the observed concentrations of DOC are considerably lower than DIC in the majority of systems examined (Figure 1). Recent succession studies in Fe(III)-oxide microbial mats (Macur et al., 2004; Beam et al., 2016b) show that autotrophic organisms are early colonizers followed by increases in heterotrophs at later successional stages. These observations are consistent with the large fractions of DIC-derived microbial C observed in numerous Fe(III)-oxide mat systems (Figure 3). Higher concentrations of DOC were observed in Washburn Spring (1.0 mM, Table 1), and may explain the lower abundance of candidate autotrophs (~4%, Table 2) in sediments present at this site. Moreover, a significant fraction of the total microbial C in these sediments is not of DIC-origin (Figure 2). The low-pH elemental sulfur system at Crater Hills also contained a significant amount of DOC (~0.2 mM), and this may contribute to the lower apparent DIC contribution to microbial C at this site.
High-temperature communities across a wide range of geochemical conditions (e.g., sulfur sediments, Fe(III)-oxide mats, filamentous streamers) exhibited large fractions of DIC-derived microbial C. Although autotrophic organisms were present in all sites that exhibited large fractions of DIC-derived microbial C, the abundance of primary autotrophs in these systems does not necessarily correlate with the minimum fractions of DIC-derived C established using 13C mixing models. For example, the relative abundance of autotrophic microorganisms in sites exhibiting >90% DIC-derived C ranged from ~17 to 93% (Table 2). Consequently, other processes should be considered when interpreting the extent of DIC-derived microbial C vs. the relative abundance of primary autotrophs. Specifically, the relative growth and/or turnover rates of autotrophic vs. heterotrophic microorganisms may not be consistent with relative abundances based on DNA sequence analysis. Viral predation likely impacts community members differentially, and could be one of the more important factors explaining lower autotroph relative abundances in communities with large fractions of DIC-derived microbial C (i.e., >50%). Future work on the composition and microbial utilization of OC sources, the relative turnover rates of autotrophs vs. heterotrophs, and the activity of different community members (e.g., transcriptomics) will assist in defining the causal factors responsible for C cycling across these geochemically diverse systems.
RJ: Conceived and co-designed experiments. Conducted bioinformatic analysis of CO2 fixation pathways. Analyzed carbon concentration and isotope data. Drafted and edited manuscript at all stages. JM, ZJ, JB, LW, MK, HK: Data collection and analysis. WI: Conceived and designed experiments. Data collection and analysis. Manuscript drafting and editing at all stages.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors acknowledge support from the DOE-Pacific Northwest National Laboratory (subcontracts 112443 and 254840), the Department of Energy (DOE)-Joint Genome Institute Community Sequencing Program (CSP 787081, CSP701), and the National Science Foundation IGERT Program for support to RJ, JB, and ZJ (NSF DGE 0654336). Work conducted by the Pacific Northwest National Laboratory (Foundational Scientific Focus Area) and the Joint Genome Institute (DOE-AC02-05CH11231) is supported by the Genomic Science Program, Office of Biological and Environmental Research, U.S. DOE. The authors appreciate assistance from C Carey and A Mazurie (MSU) for Illumina metagenome and iTag data processing, and thank C Hendrix, S Sigler and D Hallac (Center for Resources, YNP) for permitting this work in YNP (permits YELL-SCI-5068 and -5686).
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2017.00088/full#supplementary-material
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Keywords: autotrophy, CO2 fixation, stable C isotopes, geothermal, Aquificales, Crenarchaeota
Citation: Jennings RdeM, Moran JJ, Jay ZJ, Beam JP, Whitmore LM, Kozubal MA, Kreuzer HW and Inskeep WP (2017) Integration of Metagenomic and Stable Carbon Isotope Evidence Reveals the Extent and Mechanisms of Carbon Dioxide Fixation in High-Temperature Microbial Communities. Front. Microbiol. 8:88. doi: 10.3389/fmicb.2017.00088
Received: 29 July 2016; Accepted: 12 January 2017;
Published: 03 February 2017.
Edited by:Kelly Wrighton, Ohio State University, USA
Reviewed by:M. J. L. Coolen, Curtin University, Australia
Alexander V. Lebedinsky, Winogradsky Institute of Microbiology, Russia
Copyright © 2017 Jennings, Moran, Jay, Beam, Whitmore, Kozubal, Kreuzer and Inskeep. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
†Present Address: Ryan de Montmollin Jennings, Mercer University, Macon, GA
Jacob P. Beam, Bigelow Laboratory for Ocean Sciences, East Boothbay, ME


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WHAT TO DO IN SEATTLE IN 3 DAYS – ITINERARY
This 3-day Seattle itinerary helps you plan your trip. Find out about the best things to do in 3 days in Seattle – what to see, the best attractions, and more travel tips for your trip.
TRAVEL TIPS FOR YOUR 3 DAYS IN SEATTLE
If you love art, music, and a general funky vibe, Seattle is a great spot to go to. There are tons to do and see in the city, but everything is relatively close together, so you can easily make Seattle a fun 3-day trip.
If you have more time, I’d say to combine your Seattle visit with exploring more of Washington state and the West Coast. Just outside the city lies some gorgeous natural beauty worth exploring as well. It’s a unique place merging urban life and nature, so you can definitely have the best of both worlds on your Seattle trip!
DISCLAIMER: THIS POST CONTAINS AFFILIATE LINKS. THIS MEANS I MIGHT EARN A SMALL COMMISSION WHEN YOU BUY A PRODUCT/SERVICE (AT NO EXTRA COST TO YOU). MORE ABOUT IT HERE.
How to Get Around Seattle for 3 Days
- There is no need for a car in the city – parking prices are high and traffic can be crazy. For day trips – e.g. to Mount Rainier National Park – a car might be a good option. But you will not need a car for the duration of the 3 days.
- There is plenty of public transportation – including Seattle Streetcars or Seattle Center Monorail.
- If you want to get around via public transportation, you could get an All-Day Regional Transit Pass for $8 and use it on all of Seattle’s public transportation services except for the monorail and on Washington State Ferries. However, you have to buy an ORCA card first (a reusable transit card that costs $5 and can be refilled) so you can buy the $8 all-day pass from a vending machine.
- Seattle is a bike-friendly city and you can rent bikes (via the Uber or Lime App).
- Another easy way to get around is via taxis and Uber.
Best Time to Visit Seattle
You can visit Seattle year-round, but going in the spring/summer months would be the best weather-wise.
- I went in late May and found it perfect. June is also a good time as it is September and early October. The weather is pleasant and during these months prices for accommodation drop compared to the summer months.
- Summer itself is high season, and the city attracts many visitors. So accommodation prices will rise, and it gets busier.
- From late October to early May it might be very rainy and cold. Seattle is not the best destination then.
Bring layers as the weather can fluctuate. The city can get windy and rainy at times, too.
Where to Stay For 3 Days in Seattle
I would recommend staying in the downtown or Belltown neighborhoods to walk and explore the area easily. Anywhere near the Pike Place Market is a great spot and not too far from the Space Needle, Museum of Pop Culture, and the Chihuly glass garden (all must-sees).
- Some of my favorite options are the Ace Hotel and the Fairmont Olympic
- If you’re on a budget, The Best Western Pioneer Square Downtown has a good location.
Tips for Solo Female / Security
- The city, like any other, has some rougher areas. There is also a considerable population of homeless, which is usual in many US major cities. During my visit, most appeared to be drug users or mentally disturbed individuals who would shout at us.
- This is something to be aware of. I recommend just continuing along your way and ignoring any such behavior. Also, do not walk around alone at night. I walked everywhere during the day with no problems, but suggest using taxi services or Uber after dark.
Costs of Visiting Seattle in 3 Days
- Seattle is pricey! Most US cities have higher costs, but I was surprised mainly at the high cost of entry for museums and art exhibits.
- I’m used to entry on a donation basis from many places in NYC, and DC, so I was not expecting to drop $30 per person every time we went into a place, so it added up quickly!
- Be better prepared than me and bring some extra funds for sightseeing.
- Tap water is drinkable. You can save money by bringing a reusable water bottle and some pre-packaged snacks on your trip.
Restaurants to Visit For Your 3-Day Seattle Itinerary
- One of my favorite stops was The Biscuit Bitch for breakfast. It was so good I had breakfast there twice. Plus, it was a lot of fun. There’s barely any seating, so it is best to get it to go. Since it’s a hot spot, you need to get there at opening time. There will most likely still be a line, but they move very fast, and you usually don’t wait too long. Their biscuits are delicious and are served in various ways- with gravy, breakfast sandwiches, or various toppings. They offer vegetarian, vegan, and gluten-free options, too, so something for everyone, plus a coffee bar.
- Another recommendation would be Pike Place Market. It’s an awesome spot to explore and filled with various dining options and fresh goods.
BEST THINGS TO DO IN SEATTLE
So, now let’s talk about the best things to do in 3 days in Seattle.
DAY 1 OF SEATTLE ITINERARY
Day 1 in Seattle starts with the Pike Place Market.
Pike Place Market
I recommend trying to get a flight that lands you there in the morning, so you can still have some time to explore on your first day in Seattle.
After arriving and getting settled, I recommend heading to the Pike Place Market as it is one of the best places to visit in 3 days in Seattle. Pike Place Market is an iconic marketplace that started in 1907 and is the oldest continuously operating farmers’ market in the US.
- Pike Place includes a farmer’s market, a crafts market, and an array of more than 500 shops and restaurants well worth exploring.
- It attracts more than 10 million visitors annually – so you can imagine how busy it gets.
- Start your Pike Place visit with a coffee from the first-ever Starbucks before exploring everything Pike Place has to offer.
- Beware, it gets crowded! I recommend heading there earlier rather than later.
- You can even book a Pike Place Market tour and learn more from a local guide.
- If you are traveling to Seattle with kids, you might want to visit the Seattle Aquarium, located close by.
- You have free Wi-Fi at Pike Place Market!
Gum Wall in Seattle
Just near the Pike Place Market is also the iconic gum wall. It’s an alleyway in Post Alley under Pike Place Market completely covered in chewing gum. You can smell the gum (with a typical gum scent like peppermint), and yes, maybe it is a little gross but also awesome. It is worth visiting for a few photos, and this is definitely a must-see in Seattle in 3 days.
- The gum wall is a sign of Seattle’s still thriving grunge scene and was created in 1993.
- In 2015 the alleyway was scraped clean.
- It took 130 hours to remove the 2,350 pounds of gum. But it didn’t take long for Seattle natives and tourists to build it back up again.
Pioneer Square is another must-see in 3 days in Seattle and a popular Instagram place in Seattle. After all the walking, I’d recommend a stop for lunch at Piroshky Piroshky before heading to Pioneer Square with a nice walk along the waterfront.
- Pioneer Square is in the southwest of Seattle Downtown and was the heart of the city once and is especially known for its Renaissance Revival architecture.
- Pioneer Square has been around since 1852 and has several stops worth visiting, including the waterfall garden, the Klondike Goldrush Historical Park, or the Tlingit Totem Pole.
- You can also visit Seattle’s oldest skyscraper: Smith Tower, which is also in the Pioneer Square neighborhood. Make your way up to the 35th-floor speakeasy-style Observatory Bar.
- You can choose to do an underground tour which takes place in the subterranean passages at Pioneer Square. These passages were created in 1889 after Seattle’s Great Fire when the city was rebuilt over the ruins- they are known to have paranormal activity and are brimming with the history of the city.
When you’re ready for dinner, head back towards Pike Place for some good food and locally brewed beer at the Pike Pub.
DAY 2 IN SEATTLE
This is the day when you wake up early and get to it!
First, stop at the Biscuit Bitch by opening time (8 am) for an amazingly delicious breakfast and fun experience. There will be a line, but the line moves quickly, and it’s worth it.
The Space Needle
Afterward, head to The Space Needle. You’ll want to stop here first before the lines get long. After getting your ticket and traveling to the top for amazing views, you should visit 2 of my favorites – Mo Pop and Chihuly Glass Garden. All 3 places are right next to each other.
- It was built for the 1962 World’s Fair – the Century 21 Exposition “The Age of Space” so the design was perfect.
- The Space Needle is 605 feet tall and an amazing vantage point for views of Seattle. It’s a must-do on anyone’s first trip to Seattle and the ultimate icon of the Seattle skyline with glass walls and floors.
- It also includes an upper-level outdoor observation deck with open-air glass walls and Skyriser glass benches.
- Prices range between $32.50 and $37.50 (adults) and with that ticket, you get a trip to the top of the Space Needle and access to two floors of experiences at the top.
- Plan around 1,5 – 2 hours for this attraction.
Museum of Pop Culture
Mo Pop (Museum of Pop Culture) is another unique spot to add to your 3-day Seattle itinerary. It is a non-profit museum dedicated to contemporary popular culture.
- It showcases the musical history, rock stars, and of course Seattle’s own grunge subculture. It’s a fun and interactive museum unlike any other.
- They have fun and unique pop culture on display such as the “Horror” or “Sci-fi” features I visited during my stay and plenty of rotating features as well.
- Plan 1,5-3 hours for the museum and there is an entrance fee of $30 (adults).
Chihuly Glass Garden
The neighboring Chihuly Glass Garden is a very different side of Seattle’s art scene featuring elegant glass creations indoors and outdoors by world-renowned artist Dale Chihuly.
- It opened in May 2012 at the former site of the defunct Fun Forest amusement park and even those who aren’t art lovers will still appreciate the unique sculptures and beautiful gardens.
- Plan at least 1.5 hours for this attraction and the entrance fee is around $32 (adults).
All 3 stops should take up most of your day, and at some point in between, you’ll get hungry. A good local spot to grab lunch nearby would be the Skillet Counter.
Olympic Sculpture Park
While in this area, be sure to walk through the Olympic Sculpture Park as well. It’s a lovely walk right on the waterfront. The park opened in 2007 and comprises 9 acres of sculptures on display by the Seattle Art Museum (which is also nearby if you have some spare time).
- Good news: To enjoy the outdoor sculpture park with both permanent outdoor sculpture, temporary works, and site-specific installations you do not have to pay an entrance fee.
When you’re ready for dinner, head back downtown and grab a meal at Lola’s for some delicious Mediterranean fare.
If you’re a grunge music fan, grab an Uber out to Viretta Park to see the memorial bench for Kurt Cobain and his old home.
DAY 3 IN SEATTLE
Although Seattle is a big city, it is surrounded by big nature! So, whether you want to hike near Seattle or do other activities, I highly recommend on one of your days to book a day tour out of the city.
San Juan Island
My recommendation would be San Juan Island. It has a completely different feel than the city.
- Laidback island life, lighthouses, vineyards, and a charming coastal town to stroll through will give a nice break from the city’s hustle and bustle.
- You can also do whale watching! Furthermore, sea lions approach the beaches, and you can spot deer and foxes.
- If you’d rather enjoy some active time, choose a kayaking tour, rent a bike, or hike to the top of Mt. Young.
- However, plan in 2-3 hours of driving time (one way). A rental car, in this case, would be a good idea. Alternatively, there are also ferries.
- To visit San Juan Island, you need to start early or probably get there in the evening, so you can start exploring San Juan Island in the morning.
Mt. Rainier National Park
Another option is a day trip to Mt. Rainier National Park. This is a perfect choice if you enjoy hiking and wildlife spotting.
- Mt. Rainer is a glaciated volcanic peak soaring over 14,000 feet.
- It’s a gorgeous sight even from afar. If you visit over spring, you’re likely to see the slopes covered in wildflowers and rivers rushing by from melted snow.
- There are plenty of hikes: easy and panoramic ones, as well as hard ones which include climbing. The Reflection Lakes make for wonderful pictures.
- I recommend getting there early, as the parking lot fills up quickly.
- Getting here from Seattle takes about 1,5-2 hours by car.
If you are looking for something a bit more mellow, but still want some glimpses of the naturally beautiful surroundings, a day trip to Snoqualmie Falls would be a great option. Because it usually includes some vineyard visits for wine tasting.
- The 270-foot waterfall is absolutely gorgeous and doesn’t require any strenuous hiking to get to. There are upper and lower observation decks, accessible free of charge; in the evening, lights illuminate the falls.
- Nearby are several vineyards where you can taste, sample, and sip the locally-made Washington state wines.
- There is also the Northwest Railway Museum nearby.
- If you are more into some thrilling activities, you can book with companies that organize paragliding over the wonderful scenery.
- This day trip from Seattle is probably the easiest. It just takes about 30-45 minutes by car from Seattle.
No matter your style, there’s an option for everyone!
Most of these tours take up a full day, so when you get back to the city, head out to grab your last dinner in Seattle. I recommend El Borrachos. It is a tasty authentic-style Mexican spot by Pike Place with tons of options, including a vegetarian and vegan menu.
Final Thoughts on Visiting Seattle For 3 Days
Seattle is a very unique city – and there is a lot to do and see. Hopefully, this 3-day Seattle itinerary has helped you plan your trip to the city and learn how to spend 3 days in this fun city!
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About the Author
Cassandra is a US-female travel addict who loves traveling the world and works as a tour operator. She is a freelance writer for Arzo Travels, where she shares her expertise and has a special love affair with Mexico – so she knows pretty well what she is recommending to you.
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domain: travel


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Considering a river cruise abroad?
The editors of Insight Guides have just published a new book called “Great River Cruises: Europe & the Nile” ($23.95), and they’ve put together a list of the top 10 places to take a river cruise. They are:
–The Wachau Valley, a stretch of the Danube River in Austria.
–The Danube Bend, where the river runs past the Borzsony and Cserhat Hills to the north of Budapest, Hungary.
–The Iron Gates, a gorge (also on the Danube) between Serbia and Romania.
–The Rhine between Mainz and Koblenz, Germany.
–The Elbe, south of Dresden, Germany.
–The Rhone, from Vienne to Tournon in southern France.
–The Douro, from Peso da Regua to Pinhao in northern Portugal.
–The Caledonian Canal and Loch Ness, part of the Great Glen bisecting Scotland amid the Highlands.
–Kizhi Island, in Lake Onega, a stop along a popular Russian cruise itinerary following various waterways from Moscow to St. Petersburg, including the Kovzha River, which flows into the lake.
–The Nile between Aswan and Luxor in Upper Egypt.

domain: geography


Remember these are the people that complain about the hate in our hearts. When I first saw this video my emotion was pity and sadness over how society and in particular their parents had failed these young people. First you have the young man ? who is obviously bereft of a father figure to provide guidance and aid him in growing into a man. He has neither the confidence in his cause or his ability to make his case that he strikes a woman and feels justified doing so because she disagrees with him.
Then there is the young woman, in the yellow cap. Where did things go wrong for her ? What can explain that kind of hatred. She is positively gleeful in perjuring herself. You see a complete lack of moral education here. It’s like watching a feral child that has someone picked up the accoutrements civilization, (clothing, language, how to navigate a city) without any of the spiritual values that allowed the civilization to be built.
Hillary’s Tax Plan Will Kill 697,000 Jobs, Says The Tax Foundation
Lets face it, no regular readers of this site had any real hopes for it, but it’s always nice to have detailed analysis from the experts
- Hillary Clinton would enact a number of policies that would raise taxes on individual income, increase tax credits, and reform business taxation.
- According to the Tax Foundation’s Taxes and Growth Model, the plan would increase federal tax revenue by $1.4 trillion over the next decade on a static basis.
- The plan would increase marginal tax rates on individuals and businesses, which would lead to a 2.6 percent lower level of GDP. The smaller long-run economy would also lead to lower levels of wages and full-time equivalent jobs.
- After accounting for the smaller economy and narrower tax base, the plan would increase revenue by $663 billion.
- On net, the plan makes the tax code more progressive. The plan would reduce the after-tax incomes of the top 1 percent of taxpayers by 6.6 percent but increase the after-tax income of all other income groups by at least 0.1 percent. After accounting for the smaller long-run size of the economy, all after-tax incomes would fall by at least 0.1 percent in the long run.
The usual plan from the left, more taxes, less growth, less for everyone but those that can afford to navigate ever more complicated tax laws.
Manhattan Pharmacy Decides To Charge Male Customers An Extra 7 Percent Man Tax
In one such example, a Manhattan pharmacy announced on Monday that they will be hitting their male customers with an extra seven percent “man tax”—get this—in the name of “equality.”
Jolie Alony, the owner of the SoHo pharmacy, Thompson Chemists, designed the anti-man scheme as way to “level the playing field” for allegedly victimized women, in reference to the well-debunked “gender wage gap” myth and the so-called “pink tax.” Alony also says she’s making a political statement in support of Hillary Clinton and against Donald Trump. Big surprise there.
“We thought it’d be a great idea with all the political things going on—with Clinton being such a woman and the other guy and his womanizing,” said Alony.
To me the nub of the story is that in the words of Erick Erickson, we have been made to care. We care to the point of obsession to the point of insanity to the point of self destruction. This store isn’t selling sundries, it’s vending wrath and false righteousness. It’s sad that our society has fallen so far that hate is now a product sold in drugstores.
This is what has replaced freedom in America. We are free to care about whatever the outrage of the moment is. We are free to care about pointless trivia that should never have made it into print. We are free to demonstrate our virtue by hating the detestables in the public crosshairs today. We truly have the freedom of the anthill.
Quotes of the Day
Power always thinks, that it is doing God’s service when it is violating all his laws.
Drink up That’s it for the Watercooler today. As always it’s an open thread


Carmelo Anthony reiterated that it’s nonsense if Knicks general manager Glen Grunwald makes a deal at the Feb. 21 trade deadline.
“I love this team, I love the chemistry on this team — if it ain’t broke, don’t fix it,’’ Anthony said last night at the Knicks’ charity bowling event for the Garden of Dreams Foundation. “Right now we’re tops in our division. That was one of our goals. Second in the Eastern Conference, we’ll take that right now. There’s always a lot of talk around the Knicks come trade time. We don’t need that.’’
When asked about the Iman Shumpert trade talks Sunday, Anthony said, “The Knicks shouldn’t even be talking about trades.’’
Last night, he added, “We have to focus in on just basketball. In the past, there’s been distractions on the team. Now everybody’s having so much fun out there.’’
It’s still unlikely Shumpert gets moved, but Grunwald is exploring possibilities. His ears could be even more open following Sunday’s discouraging loss to the Clippers, who looked the more talented team.
According to a source, the Rockets’ director of scouting attended Sunday’s game, but he may have been checking out more than Shumpert, who may not be back to his old self until next season as he returns from ACL surgery.
The Knicks listened to a Phoenix offer in which they would receive Jared Dudley and a first-round pick. The Suns have been interested in Shumpert since July, when they asked for him in a sign-and-trade package for Steve Nash. The Knicks refused to make the second-year guard available.
“I learned really quickly in this league, you let the people do their jobs,’’ Jason Kidd said. “The front office, if they think something is needed, they’ll do it.
“With the team we have right now, we feel we can compete with the best.’’
If he’s not moved, Shumpert could be headed out of the starting lineup after this weekend’s All-Star break. Anthony has become close friends with Shumpert and realizes patience is needed with him.
“The injuries like that take time,’’ Anthony said. “He knows that. We know that. He gets frustrated sometimes. I could see that. I talk to him and hear about it. Most important is that we got guys who will help him through it. We know that’s going to take some time. We just need him to give us what he can give us at this point.’’
Coach Mike Woodson will have plenty to think about on his upcoming Caribbean weekend. The biggest question is how to get Shumpert back to his rookie-season form.
Woodson has said he has options with his starting lineup if the Raymond Felton-Shumpert-Kidd troika doesn’t work. One of them is removing Shumpert in favor of veteran swingman Ronnie Brewer, who started in the small ball alignment in November and December when the Knicks were at their best. Brewer slumped with his 3-point shooting after a fast start and it affected his defense. Eventually, Brewer wound up out of the rotation.
Physically, he doesn’t look the same on either end and Woodson admitted he’s not as mobile or quick post-surgery.
So far Woodson is being extra patient with Shumpert, but Sunday’s critical loss to the Clippers exposed the Knicks as perhaps no longer a serious title contender. Shumpert’s return was a key in whether the Knicks could dethrone the Heat.
“His lateral movement is not where I think it should be,’’ Woodson said. “His timing is off a bit. He’s not playing big minutes. He’s still on restrictive minutes. He’ll be fine. When it counts, he’ll be there.’’
An elite defender as a rookie, he tore his ACL in the first game of the Miami playoff series and it was a big reason the Knicks weren’t competitive. He could be the X-factor in another Miami series as the perimeter defender on Dwyane Wade or LeBron James.
Shumpert has been a disaster offensively, too, going scoreless in Minnesota on Friday and scoring two points vs. the Clippers. Shumpert said adjusting to small forward and being left in the deep corner for 3-pointers has been tough.
“He’s not getting into the flow and it’s just not about his offense,’’ Woodson said. “We didn’t rely on Iman scoring a lot of points last year. He’s still a rookie still trying to figure out this league.”


Benefits of Selecting Career Path Concerning the Manufacturing Industry
Since with time the technology is advancing, some career paths are no more. On the other hand, the manufacturing industry is not affected by the advancement of technology. You can find more info concerning the benefits people in manufacturing industry reap. Hence, this article needs to be read, for you to know whether a career in the manufacturing industry is for you.
The industrial manufacturing career develops always. Manufacturing career is diverse in many sectors, for instance, the aerospace, pharmaceuticals, the food and beverages manufacturing. Considering your passionate you can choose the best manufacturing sector for you. The level of technology of manufacturing companies elevate, and this makes it possible for these companies to produce different items from time to time. For instance, when it comes to food manufacturing companies, the food produces are produced different whereby some minerals vitamins and even different ingredients are added for them to be beneficial to human bodies. Hence, you should contemplate joining the manufacturing career if at all you need a developing career.
A manufacturing career helps because it creates a tangible item. Whenever someone creates something you get it is a great feeling. Therefore, in manufacturing career you can create something that you can show to the world. Whenever you create something which can be seen by people is a great experience compared to being involved in IT coding whereby you develop software which people outside the IT knowledge know nothing about your hard work concerning that software.
When getting into manufacturing career you would use your passion to specialize in one area. Since the industry develops, then you have to maintain the speed. Hence, you should consider getting online classes regarding the manufacturing sector you have specialized in. Therefore, you get to grow career wise. Again, being in the manufacturing industry does not mean that you will be dealing with the creation of products directly. You may choose to be a salesperson in a certain manufacturing company. You can be in a human resource department.
The products you manufacture on your company may be sold to local and international markets. The economy of your country and other countries may be impacted by the products you produce in manufacturing career path. Therefore, a manufacturing career is known for providing a product which is beneficial to people. However, whenever you are running your business you have to know the money saving tricks, and thus, you should contemplate on plastic conveyor.
Hence, if you are looking for options, you have in career opportunities whereby you would grow, produce an item which can be seen and touched and even something which can support the local and global economy, you need to choose the manufacturing industry.
Partner post: visit this site right here


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Hi, Byrdie readers! I’ve recently come back from a work trip to Sydney, Australia, and it got me thinking about how easy it can be to derail a healthy lifestyle when travelling or on holiday. Luckily, it’s just as easy to stay on track and still have fun when you’re abroad. You just need to keep in mind some of these tips and tricks.
It all starts on the plane! Set your intention here and now. In-flight meals tend to be high in salt and fat; let the airline know in advance if you have any dietary requirements so you can avoid gluten or dairy, for instance.
On short-haul flights, I take my own food on the plane. Aircraft cabins can be really dehydrating so I’ll always buy a large bottle of water and a big fruit salad in departures and take them on board. Of course, if you’re travelling long-haul you can’t not eat! I take sachets of WelleCo Nourishing Protein (£28) and a shaker with me on the flight, as you can mix it with water, and then I try to choose the healthiest options on the menu.
I’ll apply Elizabeth Arden 8-Hour Cream (£26) to my lips and be sure to mist my skin throughout the whole journey. I find it hard to relax in general, but This Works Deep Sleep Pillow Spray (£16) misted onto a travel pillow or on scarf can be really calming (I’m not a big fan of flying!). I always have an eye mask and ear plugs in my carry-on too.
Flying causes stress on the body, and while it is fun to have a glass of champagne or wine on the flight, I try to limit that, especially if travelling for work. While it’s not proven, I always feel like one drink in the air is equivalent to about four on land! I don’t want to be a party pooper, but I don’t want to arrive hungover either!
You don’t need to go to a class, there are plenty of workouts you can do in your hotel room like yoga, Kayla Itsines Bikini Body Guides or Jillian Michaels’s workouts (I love her 6 Weeks Six-Pack Abs workout), or I’ll head to the beach and exercise using just my body weight.
Walking, jogging or hiking are great ways to explore an area while getting fit.
If I’m travelling to visit a friend, I’ll get them to recommend a workout. One of my friends suggested a really amazing place in NYC recently called The Class where the session consisted of primal-type moves which was fun. I’m also a big fan of things like ClassPass where you sign-up and book in for workouts anywhere in the world.
In my suitcase I’ll pack some gym kits, lightweight trainers and some resistance bands. You could even pack a TRX and wrap it around a palm tree!
You can wear this on the beach because it's water resistant and it has antioxidant vitamin E, which helps protect your skin in the sun.
Jojoba oil is great for skin irritations and breakouts. At night, after being in the sun all day, my skin can feel quite dehydrated so I apply this oil.
I love this for getting that beachy looking hair at home and on holiday.
How do you stay fit and healthy on holiday? Share your tips in the comment box below.

domain: health


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Investigated Ducks kicked off team
Columbia Daily Tribune
Three Oregon basketball players, who were investigated but not charged in an alleged sexual assault, have been dismissed from the team.
Damyean Dotson, 19, Dominic Artis, 19 and Brandon Austin, 18, were suspended after the school received a police report concerning allegations made by a student who said she was assaulted by the players in early March. School officials announced yesterday that the players would not return to the Ducks because of conduct that is not befitting of student athletes.
The Lane County district attorney’s office investigated the allegations and determined that there was not enough evidence to prosecute the players on criminal charges.

domain: sociology


The Force Awakens's John Boyega Walked the Star Wars Celebration Floor in Disguise
What do you do if you're at Star Wars Celebration as both talent and a fan? You walk the floor incognito, of course! Star Wars: The Force Awakens star John Boyega walked the floor dressed as - what else? - a Stormtrooper, successfully hiding his identity from the unsuspecting fans who would have loved to swarm him:
A photo posted by @john_boyega on
I hope whoever this fan is recognizes himself in this picture. Because, you know, life made and everything.
A video posted by @john_boyega on
Boyega isn't the first to walk the floor of a convention behind the safety of a mask; Daniel Radcliffe, Peter Jackson, and who knows how many others have all bumped shoulders with unwitting fans at cons. So next time you're at a convention, remember that any number of people hiding behind masks might be your favorite celebrities.
Star Wars: The Force Awakens also stars Harrison Ford, Carrie Fisher, Mark Hamill, Daisy Ridley, Oscar Isaac, and Adam Driver. The film comes out in theaters on December 18.


Motorola's designers are really good. The company's smartphones in the middle and high price range are always beautiful and original. They manage to easily attract the attention of users of Android models. One of the exciting proposals in 2021 is the Motorola Edge 20 lite. It has good photo capabilities.
Before reading the full review, you can familiarize yourself with the detailed Motorola Edge 20 Lite specs or watch the video below the article.
- Release date: 2021, July 29
- Colors: Electric Graphite, Lagoon Green
- Dimensions: 165.9 x 76 x 8.3 mm.
- Weight: 185 g.
- Screen: 6.7" inc, 1080 x 2400, OLED
- Camera : Triple, 108MP
- Chipset: MediaTek MT6853 Dimensity 720 5G (7 nm)
- CPU: Octa-core (2x2.0 GHz Cortex-A76 & 6x2.0 GHz Cortex-A55)
- Memory: 128GB 6GB RAM, 128GB 8GB RAM
- Battery: 5000, Li-Po, non-removable
- Network: GSM / HSPA / LTE / 5G
- Operating system: Android 11
- Sensors: Fingerprint (side-mounted), accelerometer, gyro, proximity, compass
Visually, the smartphone is almost no different from modern flagships. Thin frames around the display, memorable triple camera layout and bright glass back panels with the familiar logo of the developer. All this makes Motorola Edge 20 lite memorable and attractive against the background of monotonous competitors.
The camera module is positioned as standard in the upper left part of the rear panel. It protrudes slightly above the surface.
With its 185 grams Motorola Edge 20 lite is comfortable to carry - not too big, with nicely rounded corners. The front of its body is made of glass and the back is made of plastic. The latter is pleasant to the touch, strong enough that you do not see scratches on it for a long time. You can choose between two colors - graphite and green. Both options will appeal to young people looking for an attractive device.
The fingerprint scanner is side mounted and you will easily get used to using it. It works quickly and accurately. Reliable protection is against unfriendly eyes.
Motorola Edge 20 lite has a large enough AMOLED display with a diagonal of 6.7 inches. Its resolution is FullHD +, and the picture refresh rate is decent - 90 Hz. Another reason to praise the display is the oleophobic layer and increased brightness. It is convenient to interact with the device even in direct sunlight and in the dark.
The aspect ratio of the screen is 19.5: 9. It occupies over 87% of the front panel, which is quite a high figure for a modern smartphone. The image quality is judged by the pixel density per inch - 385. This is good for a smartphone in this price range.
The technical characteristics of Motorola Edge 20 lite will not provoke astonishment and delight among gamers. However, the performance of the Mediatek Dimensity 720 is sufficient for a bunch of resource-intensive tasks. You also have 6 or 8 GB of RAM and a Mali G57MC3 video accelerator. They help the chipset to realize good results. In other words, the Motorola Edge 20 lite is a smartphone with immodest gaming power for its price. It supports 5G networks and provides reasonable power consumption.
The internal storage is 128 GB and can hold enough collections of photos, videos and music. You can add a memory card and expand it at any time.
The smartphone comes with the Android 11 operating system at the factory.
The strongest asset of the Motorola Edge 20 lite are the cameras. The rear consists of three modules. The main one is 108-megapixel, wide-angle with an aperture of f / 1.9. It is complemented by an 8-megapixel sensor, ultra-wide angle, with which you will take impressive panoramic and group photos. The 2-megapixel module is tasked with measuring depth to have exciting bokeh photos.
The front camera is single, but wide-angle and 32 MP. With the Motorola Edge 20 lite in hand, you have every chance to make impressive videos and photos, thanks to improved algorithms. Video recording has a maximum resolution of 4K at 30 frames per second.
Although the optical stabilization is an attribute of the brand's flagship production, its absence here is not before taking flawless daily photos. The evening ones have less details and visible noises. However, they are subject to editing and you can republish them on social networks.
Under the attractive body of the model lies a battery with a capacity of 5000 mAh. It provides about 10-12 hours of active screen time in a day and a half of use.
For the convenience of the user, Motorola has provided technology for fast charging of the 30W battery. This way you will be able to use your favorite applications again and surf the Internet in negative time.
Motorola Edge 20 lite leaves a good impression at first glance. The beautiful design, complemented by an excitingly large display with a picture refresh rate of 90 Hz, further convinces you to have it. And when you learn what photo opportunities there are, you can be disarmed.
The smartphone features water protection, a fast side-mounted fingerprint scanner and 30W fast charging technology. It also comes with ample internal storage that you can expand at any time. It supports 2 SIM cards and is even likely to please gamers with its performance.
Motorola Edge 20 Lite video review
Disclaimer: The information presented in this article is based on our team's personal experience with the Motorola Edge 20 Lite and third-party sources. While every effort has been made to provide accurate and reliable information, readers should keep in mind that this is a subjective assessment. The writing of this article was not paid for or sponsored by Motorola.


text: I’m taking you back a bit to my first night in Texas. After our flight and finally checking into our hotel, we hopped in a gypsy cab downtown, and ended up right smack dab in the middle of 6th street. We STILL agree our driver was drunk. The music was loud, the crowds crazy, and I think the whole city was down there for the night. The dreadlocks. The boys and their guitars high their backs. There really was a little bit of everything roaming the streets. Bars were completely packed, and you could literally see gigs just by walking down the sidewalk. FREE LIVE MUSIC!! All everyone needed on this crazy night. I was just happy to survive the crowds to snap photos, and with my 3 giant handbags! Talk about looking like a tourist. And the HEAT! First time in months we’ve felt anything like it. Laughing and smiling because we didn’t need a stupid heavy coat. So there you have it, our first night was incredible. Right away we knew SXSW was going to be amazing. After trying our very first tex-mex taco, we were back at our hotel for a short snooze, anxiously awaiting our first full day at SXSW. I miss these nights, just before what was an inspiring adventure.


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Most quilters think of their walking foot as a tool to “stitch in the ditch”. Numerous quilting designs are achievable with this great attachment that provides a quilter with a comfortable level of control and success. Current quilting designs make use of straight lines, as well as slightly curved lines, to produce grids and patterns that provide interesting texture to your quilts. You will learn how to make the most out of your walking foot while trying out different techniques and patterns. Jacquie Gering’s book WALK is required for the class. “Basics of Machine Quilting with the Walking Foot” class is a pre-requisite for this class.
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domain: artistic