Diversity of microbial communities in hot springs of Sri Lanka as revealed by 16S rRNA gene high-throughput sequencing analysis.

Characterization of hot spring microbiota is useful as an initial platform for exploring industrially important microbes. The present study focused on characterization of microbiota in four hot springs in Sri Lanka: Maha Oya; Wahava; Madunagala; and Kivlegama using high throughput 16S amplicon sequencing. Temperatures of the selected springs were ranged from 33.7 °C to 52.4 °C, whereas pH ranged from 7.2 to 8.2. Bacteria were found to be the dominant microbial group (>99%) compared to Archaea which represented less than 1% of microbiota. Four hot springs comprised of unique microbial community structures. Proteobacteria, Firmicutes, Bacteroidetes, Cloroflexi, Deinococcus and Actenobacteria were the major bacterial phyla. Moderately thermophilic genera such as Thermodesulfobacteria and Deinococcus-Thermus were detected as major genera that could be used in industrial applications operating at temperatures around 50 °C and alkaline reaction conditions.

COMPARISON OF THE RADIO-ISOTOPIC COMPOSITION BETWEEN HOT SPRING WATER OF HAMMAM DEBAGH AND ITS ASSOCIATED DEPOSITS.

A radiological analysis of hot spring water of Hammam Debagh (also called Hammam Meskhoutine) and its associated deposits was realized. Hammam Debagh is considered to be the second hottest springs in the world with a temperature of 98°C and this lead to the creation of travertine. This later was formed because of limestone deposited by mineral springs in a rapid sedimentation of calcium, magnesium bicarbonates, and sulphides. The analysis showed that two-thirds of radioactivity was left by spring water in their deposits. The presence of uranium in the spring deposits was due to the property of the uranyl ion to form stable complexes with carbonates and precipitates. The radiation hazards indexes were estimated and compared with those given in literature.

Assessing the Committed Effective Dose From 226Ra in Thermal Spring Water From San Diego De Alcala, Chihuahua, Mexico.

The oral administration of mineral-rich spring water is known as hydropinic treatment and is used to treat certain ailments. Health benefits are attributed to thermal spring water containing radioactive elements such as radium; this has popularized use of such radioactive water in various parts of the world, causing those who ingest it to increase their internal radiation dose. The goal of this study was to assess the activity concentrations of Ra present in the thermal spring waters of San Diego de Alcala, in the state of Chihuahua, Mexico, and to estimate the health risk posed to patients by the effective dose received from ingesting this water during hydropinic treatments. Water samples were taken from different areas of the San Diego de Alcala thermal springs, and pH, temperature, electrical conductivity, and total dissolved solids were measured. The Ra activity concentrations were measured with a liquid scintillation counter. The activity concentrations of Ra in sampled water varied from 125 to 452 mBq L with an average of 276 ± 40 mBq L. The committed effective dose from each of the Ra activity concentrations found in samples ranged from 9.80 × 10 to 4.0 × 10 mSv for hydropinic treatments being carried out in San Diego de Alcala thermal spring spas. Different treatments had different intake rates (200, 600, 1,000, and 1,500 mL d) and occurred over periods of 2 or 3 wk. According to the guidelines of the US Environmental Protection Agency, the maximum permissible amount of radium in drinking water is 185 mBq L; the Ra content in most of the collected samples exceeded this limit. The committed effective doses varied with Ra concentration and intake rate; none exceeded the World Health Organization's reference dose for drinking water of 0.1 mSv y, which is the maximum amount to which the population should be exposed.

Bacterial communities of the microbial mats of Chokrak sulfide springs.

This is the comparative investigation of the composition of phototrophic microbial mats developing in sulfide-rich saline Chokrak springs with outflow at the shore of the hypersaline lake Chokrak by means of next-generation sequencing. The springs are characterized by low temperature (~ 15 °C), near-neutral pH (6.7-8.5), and high-sulfide content. In the species composition the benthic microbial communities of Chokrak springs are similar to microbial mats of marine supralittoral and lagoons. Our results showed that salinity limitation had a significant effect on the species composition of benthic microbial communities developing at the outflow of the Chokrak springs. Predominant oxygenic phototrophs belonged to the genera Phormidium, Lyngbya, Leptolyngbya, Geitlerinema, and Arthrospira. Anoxygenic phototrophic bacteria were represented by halophilic green sulfur bacteria Prosthecochloris spp., halotolerant Chlorobaculum sp., as well as marine and extremely halophilic purple bacteria Roseospira, Rhodovibrio, and Halochromatium. Monoculture of a new species of halotolerant anoxygenic filamentous phototrophic bacteria was isolated.

Natural radioactivity of thermal springs and related precipitates in Gellért Hill area, Buda Thermal Karst, Hungary.

The elevated radioactivity of the thermal waters of Buda Thermal Karst (BTK), Hungary is known and studied since the beginning of the 20th century. In the recent studies, the anomalous 222Rn/226Ra ratios have drawn the attention to the existence of local 222Rn source. Biogeochemical precipitates (i.e. biofilms) in spring caves were found to have high adsorption capacity, accumulating e.g. 226Ra. Biogeochemical precipitates are ubiquitous in the thermal springs of BTK, occurring in different amount and colours (dark grey, brown, red, white), and have different microbial communities and elemental composition. The detailed investigation of the radioactivity of spring waters highlighted the different 226Ra and 222Rn activity concentrations. The present study aimed to survey the radioactivity of the thermal springs of Gellért Hill area, together with the biogeochemical precipitates and air above the water level, and to assess the evolution of the radioactivity of known-aged precipitates, formed during in situ experiments. We found that the basic physicochemical parameters of the spring waters (field parameters, major ions) do not affect the adsorption capacity of biogeochemical precipitates. It was revealed by the conducted in situ experiments, that the flow conditions influence the evolution rate of precipitates, so their adsorption capacity. The 222Rn activity concentrations of spring waters are dependent on the area of the water surface, volume of air space above the water level, ventilation of the caves/channels and presence of calcite layer on the water surface. The latter has a blocking effect on degassing.

Radon concentration in hydrogeothermal deposit and spas of Boyacá, Colombia.

Inhaled radon and its progenies induce health concern due to high activity-concentration in selected thermal spas of Boyacá region. Hydrogeothermal water sources in a high risk seismic area, are studied to determine by water bubbling method radon concentration values; their occurrence is between few hundreds and 2000 Bq dm-3. Deposits, existing in this area, reach at the surface soil gas radon concentration up to 210 kBq m-3. Maintenance workers, health tourists and visitor's possible detrimental health effects, are discussed in relation to radon balneotherapy beneficial aspects.

RADIONUCLIDE AND MAJOR ELEMENT ANALYSIS OF THERMAL AND MINERAL WATERS IN CROATIA WITH A RELATED DOSE ASSESSMENT.

Major elements concentrations (Na, K, Ca, Mg) and the activity concentrations of 226,228Ra, 234,238U, 210Po, 210Pb, 40K and 137Cs in northern and eastern Croatian thermal and mineral waters, collected directly from springs (or wells), are presented herein with total effective doses assessed for those waters that are considered as drinking 'cures' and are available for consumption. The methods used for radionuclide determination included alpha-particle spectrometry, gas-proportional counting and gamma-ray spectrometry, while the major element composition was determined by ICP-MS. The activity concentrations of all of the radionuclides were found to be below the guidance levels set by the WHO and EC Directive, with the exception of one water sample that measured 0.26 Bq L-1 of 228Ra. The effective ingestion dose assessment for the consumption of the so-called water 'cures' during 1, 2 or 4 weeks' time period throughout 1 year was well below the recommended 0.1 mSv for drinking water.

Study of isotopes of carbon, thorium, and uranium in travertine and thermal spring samples: implications for effects of changes in geochemical environment and processes.

Data on modern radiocarbon activity in the old travertine formations of the Pymvashor hydrothermal system were used, in combination with 230Th/U dating of the travertine, to estimate the 14C contents of the total dissolved inorganic carbon in the ancient thermal water at the time of precipitation of the travertine (14Ccalc). With the known values of 14Ccalc and average age of the thermal water, and under the assumption that the residence time of the water in aquifer was constant over the last 13.9 ± 1.5 ka, the initial 14C contents (14C0) in the ancient thermal water were estimated. The findings in this study are that (1) both 14C0 and δ13С have decreased in young waters compared to the ancient waters; (2) although atmospheric 14C activity (14Catm) has also decreased in the same time, the decrease in 14C0 is faster than the decrease in 14Catm. Under certain assumptions, one could link changes in 14C0 and δ13C to climate change. Decrease in δ13С of soil CO2 and decrease in 14C0 was caused predominantly by warmer and wetter climate, decomposition of fossil organic matter, and decrease in 14Catm. There could be also increased dissolution of solid carbonate and increased carbon exchange between DIC and soil CO2, caused by thawing permafrost.

Isolation of highly thermostable ß-xylosidases from a hot spring soil microbial community using a metagenomic approach.

The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable ß-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of >500 bp in length. Similarity search for ß-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for ß-xylosidase activity. A total of 82 proteins exhibited ß-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.

Oxygenic and anoxygenic photosynthesis in a microbial mat from an anoxic and sulfidic spring.

Oxygenic and anoxygenic photosynthesis were studied with microsensors in microbial mats found at 9-10 m depth in anoxic and sulfidic water in Little Salt Spring (Florida, USA). The lake sediments were covered with a 1-2 mm thick red mat dominated by filamentous Cyanobacteria, below which Green Sulfur Bacteria (GSB, Chlorobiaceae) were highly abundant. Within 4 mm inside the mats, the incident radiation was attenuated to undetectable levels. In situ microsensor data showed both oxygenic photosynthesis in the red surface layer and light-induced sulfide dynamics up to 1 cm depth. Anoxygenic photosynthesis occurred during all daylight hours, with complete sulfide depletion around midday. Oxygenic photosynthesis was limited to 4 h per day, due to sulfide inhibition in the early morning and late afternoon. Laboratory measurements on retrieved samples showed that oxygenic photosynthesis was fully but reversibly inhibited by sulfide. In patches Fe(III) alleviated the inhibition of oxygenic photosynthesis by sulfide. GSB were resistant to oxygen and showed a low affinity to sulfide. Their light response showed saturation at very low intensities.

hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for surviving in the hot spring run-off aquatic ecosystem.

Changes in the expression of a number of hsp genes in minnow Puntius sophore collected from a hot spring run-off (Atri hot spring in Odisha, India; 20o09'N 85°18'E, 36-38 °C) were investigated to study the upper thermal acclimation response under heat stress, using same species from aquaculture ponds (water temperature 27 °C) as control. Expression of hsp genes was analyzed in both groups using RT-qPCR, which showed up-regulation of hsp90 (2.1-fold) and hsp47 (2.5-fold) in hot spring run-off fishes, whereas there was no alteration in expression of other hsps. As the fish inhabit the hot spring run-off area for very long duration, they could have adapted to the environment. To test this hypothesis, fishes collected from hot spring run-off were divided into two groups; one was heat-shocked at 41 °C/24 h, and the other was acclimatized at 27 °C/24 h. Up-regulation of all the hsps (except hsp78) was observed in the heat-shocked fishes, whereas expression of all hsps was found to be down-regulated to the basal level in fishes maintained at 27 °C/24 h. Pathway analysis showed that the expressions of all the hsps except hsp90 are regulated by the transcription factor heat shock factor 1 (Hsf1). This study showed that hsp90 and hsp47 play an important role in Puntius sophore for surviving in the high-temperature environment of the hot spring run-off. Additionally, we show that plasticity in hsp gene expression is not lost in the hot spring run-off population.

Hydrothermal gases in a shallow aquifer at Mt. Amiata, Italy: insights from stable isotopes and geochemical modelling.

We investigate the interaction between hydrothermal gases and groundwater in a major aquifer exploited for potable supply in the geothermal-volcanic area of Mt. Amiata, Central Italy. Two springs and two wells located on different sides of the volcanic edifice have been repeatedly sampled over the last 11 years. More than 160 chemical analyses and 10 isotopic analyses of total dissolved carbon (δ(13)C - total dissolved inorganic carbon (TDIC) = -15.9 to -7.8 ‰ vs. V-PDB) and sulphate (δ(34)S-SO4 = -6.9 to 5.1 ‰ vs. V-CDT) have been processed with geochemical modelling techniques. Best-fitting conditions between analytical data and model outputs have been achieved by numerical optimization, allowing for a quantitative description of gas-water-rock interactions occurring in this aquifer. Numerical calculations support a conceptual model that considers water-rock interactions to occur in the volcanic aquifer after inflow of deep-seated gases (CO2(g) and H2S(g)), and total conversion of H2S(g) to SO4, in the absence of mixing with geothermal waters from reservoirs currently exploited for electricity generation.

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous 'streamer' community.

The candidate archaeal phylum 'Aigarchaeota' contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous 'streamer' community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus 'Calditenuis aerorheumensis' for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.

Determination of arsenic speciation in sulfidic waters by Ion Chromatography Hydride-Generation Atomic Fluorescence Spectrometry (IC-HG-AFS).

A method for the analysis of arsenic species in aqueous sulfide samples is presented. The method uses an ion chromatography system connected with a Hydride-Generation Atomic Fluorescence Spectrometer (IC-HG-AFS). With this method inorganic As(III) and As(V) species in water samples can be analyzed, including arsenite (HnAs(III)O3(n-3)), thioarsenite (HnAs(III)S3(n-3)), arsenate (HnAs(V)O4(n-3)), monothioarsenate (HnAs(V)SO3(n-3)), dithioarsenate (HnAs(V)S2O2(n-3)), trithioarsenate (HnAs(V)S3O(n-3)) and tetrathioarsenate (HnAs(V)S4(n-3)). The peak identification and retention times were determined based on standard analysis of the various arsenic compounds. The analytical detection limit was ~1-3 µg L(-1) (LOD), depending on the quality of the baseline. This low detection limit makes this method also applicable to discriminate between waters meeting the drinking water standard of max. 10 µg L(-1) As, and waters that do not meet this standard. The new method was successfully applied for on-site determination of arsenic species in natural sulfidic waters, in which seven species were unambiguously identified.

Detection of hydrogen sulfide through photoluminescence quenching of penicillamine-copper nanocluster aggregates.

We have developed a one-pot, inexpensive, simple and rapid method to synthesize photoluminescent copper nanocluster (Cu NC) aggregates from Cu(2+) ions in 65% (v v(-1)) dimethylformamide aqueous solution containing penicillamine (PA) as a capping and reducing agent. As-prepared PA-Cu NC aggregates emit at 580 nm when excited at 326 nm, with a quantum yield of 2.0%. The photoluminescence of PA-Cu NC aggregates originate from ligand-to-metal charge transfer, which is supported by a long lifetime (126.5 ns) and a large Stokes shift (254 nm). As-prepared PA-Cu NC aggregates have different emission wavelengths with the same excitation wavelength in various organic-aqueous solutions. The PA-Cu NC aggregates are highly selective and sensitive to the detection of hydrogen sulfide (H2S), based on analyte-induced photoluminescence quenching through the formation of CuS nanoparticles. The probe allows the detection of H2S, with a linear range of 1-100 µM and a limit of detection (signal-to-noise ratio = 3) of 500 nM. The practicality of this probe has been validated through the analysis of hot spring water samples.

Human health risks of geothermally derived metals and other contaminants in wild-caught food.

Arsenic (As) and mercury (Hg) associated with geothermally influenced lakes and rivers represent a potential health risk to communities where wild-caught food is consumed. The Rotorua Lakes region of New Zealand has extensive natural geothermal activity and a large proportion (35%) of indigenous Maori population, for whom wild food gathering is an important cultural activity. The aim of this study was to measure selected heavy metal and organochlorine (OC) concentrations in important local fish and shellfish species and assess the potential health risk to the local population of consuming these species. Following U.S. Environmental Protection Agency (EPA) protocols, consumption limits were calculated based on both excess lifetime cancer risk and noncancer risk. These were compared with local consumption rates, which were determined by questionnaire (n = 19). Median and 95th percentile contaminant concentrations were calculated to approximate random and most extreme contaminant consumption scenarios. Only Hg concentrations exceeded established Food Standards Australia New Zealand (FSANZ) guideline values of 0.5 mg/kg, namely, for rainbow trout (Oncorhynchus mykiss; 62% of the study sites) and koura (freshwater crayfish; Paranephrops planifrons; 25% of sites). The major risk was from consumption of trout, where the local consumption rate (1.5 meals/mo) exceeded the consumption limit of 0.9 meals/mo (median data) and 0.4 meals/mo (95th percentile data). Shellfish--pipi (Paphies australis) and mussel (Perna canaliculus)--collected from the only estuarine site also had local consumption rates (3.5 meals/mo) above calculated consumption limits (2.6 and 2.9 meals/mo, respectively). Our results, while based on a limited sample size and therefore exploratory in nature, nevertheless provide the basis for developing consumption guidelines. This study makes a significant contribution to broadening our understanding of the complexities of managing customary fisheries.

Molecular analysis of the benthos microbial community in Zavarzin thermal spring (Uzon Caldera, Kamchatka, Russia).

BACKGROUND: Geothermal areas are of great interest for the study of microbial communities. The results of such investigations can be used in a variety of fields (ecology, microbiology, medicine) to answer fundamental questions, as well as those with practical benefits. Uzon caldera is located in the Uzon-Geyser depression that is situated in the centre of the Karym-Semyachin region of the East Kamchatka graben-synclinorium. The microbial communities of Zavarzin spring are well studied; however, its benthic microbial mat has not been previously described. RESULTS: Pyrosequencing of the V3 region of the 16S rRNA gene was used to study the benthic microbial community of the Zavarzin thermal spring (Uzon Caldera, Kamchatka). The community is dominated by bacteria (>95% of all sequences), including thermophilic, chemoorganotrophic Caldiserica (33.0%) and Dictyoglomi (24.8%). The benthic community and the previously examined planktonic community of Zavarzin spring have qualitatively similar, but quantitatively different, compositions. CONCLUSIONS: In this study, we performed a metagenomic analysis of the benthic microbial mat of Zavarzin spring. We compared this benthic community to microbial communities found in the water and of an integral probe consisting of water and bottom sediments. Various phylogenetic groups of microorganisms, including potentially new ones, represent the full-fledged trophic system of Zavarzin. A thorough geochemical study of the spring was performed.

[Estimation of effective doses derived from radon in selected SPA centers that use geothermal waters based on the information of radon concentrations]. / Szacowanie dawek efektywnych pochodzicych od radonu w wybranych placówkach geotermalnych SPA na podstawie informacji o stezeniach radonu.

BACKGROUND: Geothermal waters contain, among other components, soluble radon gas. Alpha radioactive radon is a health hazard to humans, especially when it gets into the respiratory tract. SPA facilities that use geothermal water can be a source of an increased radiation dose to people who stay there. Based on the available literature concerning radon concentrations, we assessed exposure to radon among people - workers and visitors of Spa centers that use geothermal waters. MATERIAL AND METHODS: Radon concentrations were analyzed in 17 geothermal centers: in Greece (3 centers), Iran (5), China (4) and India (5). Doses recived by people in the SPA were estimated using the formula that 1 hour exposure to 1 Bq/m3 of radon concentration and equilibrium factor F = 0.4 corresponds to an effective dose of 3.2 nSv. RESULTS: We have found that radon levels in SPAs are from a few to several times higher than those in confined spaces, where geothermal waters are not used (e.g., residential buildings). In 82% of the analyzed SPAs, workers may receive doses above 1 mSv/year. According to the relevant Polish regulations, people receiving doses higher than 1 mSv/year are included in category B of radiation exposure and require regular dosimetric monitoring. Doses received by SPA visitors are much lower because the time of their exposure to radon released from geothermal water is rather short. CONCLUSIONS: The analysis of radon concentration in SPA facilities shows that the radiological protection of people working with geothermal waters plays an important role. It seems reasonable to include SPA workers staying close to geotermal waters into a dosimetric monitoring program.

Division-specific differences in bacterial community assembly of two alkaline hot spring ecosystems from Yellowstone National Park.

A fundamental issue in ecology is whether communities are random assemblages or, alternatively, whether there are rules that determine which combinations of taxa can co-occur. For microbial systems, in particular, the question of whether taxonomic groups exhibit differences in community organization remains unresolved but is critical for our understanding of community structure and function. Here, we used presence-absence matrices derived from bar-coded pyrosequencing data to evaluate the assembly patterns of eight bacterial divisions distributed along two Yellowstone National Park hot spring outflow channels. Four divisions (Cyanobacteria, Chloroflexi, Acidobacteria, and Cytophaga-Flavobacterium-Bacteroides) exhibited less co-occurrence than expected by chance, with phototrophic taxa showing the strongest evidence for nonrandom community structure. We propose that both differences in environmental tolerance and competitive interactions within divisions contribute to these nonrandom assembly patterns. The higher degree of nonrandom structure observed for phototrophic taxa compared with the other divisions may be due in part to greater overlap in resource usage, as has been previously proposed for plant communities.

Biogeochemical characteristics of Kuan-Tzu-Ling, Chung-Lun and Bao-Lai hot springs in southern Taiwan.

Hot springs are the important natural sources of geothermally heated groundwater from the Earth's crust. Kuan-Tzu-Ling (KTL), Chung-Lun (CL) and Bao-Lai (BL) are well-known hot springs in southern Taiwan. Fluid and mud (sediments) samples were collected from the eruption points of three hot springs for detailed biogeochemical characterization. The fluid sample displays relatively high concentrations of Na(+) and Cl(-) compared with K(+), Mg(2+), Ca(2+), NO(2) (-), and SO(4) (2-), suggesting a possible marine origin. The concentrations of Fe, Cr, Mn, Ni, V and Zn were significantly higher in the mud sediments compared with fluids, whereas high concentrations of As, Ba, Cu, Se, Sr and Rb were observed in the fluids. This suggests that electronegative elements were released during sediment-water interactions. High As concentration in the fluids was observed to be associated with low redox (Eh) conditions. The FTIR spectra of the humic acid fractions of the sediments showed the presence of possible functional groups of secondary amines, ureas, urethanesm (amide), and silicon. The sulfate-reducing deltaproteobacterium 99% similar to Desulfovibrio psychrotolerans (GU329907) were rich in the CL hot spring while mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium that 99% similar to Clostridium sulfidigenes (GU329908) were rich in the BL hot spring.