Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

Bacterial diversity in five Icelandic geothermal waters: temperature and sinter growth rate effects

The microbial ecology associated with siliceous sinters was studied in five geochemically diverse Icelandic geothermal systems. Bacterial 16S rRNA clone libraries were constructed from water-saturated precipitates from each site resulting in a total of 342 bacterial clone sequences and 43 species level phylotypes. In near-neutral, saline (2.6–4.7% salinity) geothermal waters where sinter growth varied between 10 and ~300 kg year⁻¹ m⁻², 16S rRNA gene analyses revealed very low (no OTUs could be detected) to medium (9 OTUs) microbial activity. The most dominant phylotypes found in these waters belong to marine genera of the Proteobacteria. In contrast, in alkaline (pH = 9–10), meteoric geothermal waters with temperature = 66–96°C and <1–20 kg year⁻¹m⁻² sinter growth, extensive biofilms (a total of 34 OTUs) were observed within the waters and these were dominated by members of the class Aquificae (mostly related to Thermocrinis), Deinococci (Thermus species) as well as Proteobacteria. The observed phylogenetic diversity (i.e., number and composition of detected OTUs) is argued to be related to the physico-chemical regime prevalent in the studied geothermal waters; alkaliphilic thermophilic microbial communities with phylotypes related to heterotrophic and autotrophic microorganisms developed in alkaline high temperature waters, whereas halophilic mesophilic communities dominated coastal geothermal waters.     

Exponential growth of “snow molds” at sub-zero temperatures: an explanation for high beneath-snow respiration rates and Q 10 values

Numerous studies have demonstrated exceptionally high temperature sensitivity of the beneath-snow respiratory flux in cold-winter ecosystems. The most common, but still untested, explanation for this high sensitivity is a physical one based on the observation that water availability in soils increases exponentially as soils warm from −3 to 0°C. Here, we present evidence for a biological hypothesis to explain exponential kinetics and high Q ₁₀ values as beneath-snow soils warm from −3 to 0°C during the early spring in a high-elevation subalpine forest. First, we show that some of the dominant organisms of the beneath-snow microbial community, “snow molds”, exhibit robust exponential growth at temperatures from −3 to −0.3°C. Second, Q ₁₀ values based on growth rates across the temperature range of −2 to −0.3°C for these snow molds vary from 22 to 330. Third, we derive an analytical equation that combines the relative contributions of microbial growth and microbial metabolism to the temperature sensitivity of respiration. Finally, we use this equation to show that with only moderate snow mold growth (several generations), the combined sensitivities of growth and metabolism to small changes in beneath-snow soil temperature, create a double exponential in the Q ₁₀ function that may explain the extremely high (~1 × 10⁶) Q ₁₀ values observed in past studies. Our biological explanation for high Q ₁₀ levels is supported by several independent studies that have demonstrated build up of microbial biomass under the snow as temperatures warm from −2 to 0°C.     

Dehydration and cold hardiness in the Arctic Collembolan Onychiurus arcticus Tullberg 1876

Specimens of the Arctic Collembolon Onychiurus arcticus were exposed to desiccation at several subzero temperatures over ice and at 0.5 °C over NaCl solutions. The effects of desiccation on water content (WC), body fluid melting point (MP), supercooling point (SCP) and survival were studied at several acclimation temperatures and relative humidities. Exposure to temperatures down to −19.5 °C caused a substantial and increasing dehydration. At the lowest exposure temperature unfrozen individuals lost 91.6% of the WC at full hydration but more than 80% of the individuals survived when rehydrated. Exposure at 0.5 °C to decreasing relative humidities (RH) from 100% to 91.3% caused increasing dehydration and increasing mortality. Survival of equally dehydrated individuals was higher at subzero temperatures than at 0.5 °C. Concurrent with the decline in WC a lowering of the MP was observed. Animals exposed to −3 °C and −6 °C over ice for 31 days had a MP of −3.8 and < −7.5 °C, respectively. Specimens from a laboratory culture had a mean SCP of −6.1 °C, and acclimation at 0 or −3 °C had little effect on SCPs. Exposure at −8.2 °C over ice for 8 days, however, caused the mean SCP to decline to −21.8 °C due to the severe dehydration of these individuals. Dehydration at 0.5 °C in 95.1 and 93.3% RH also caused a decline in SCPs to about −18 °C. Individuals that had been acclimated over ice at −12.4 °C or at lower temperatures apparently did not freeze at all when cooled to −30 °C, probably because all freezeable water had been lost. These results show that O. arcticus will inevitably undergo dehydration when exposed to subzero temperatures in its natural frozen habitat. Consequently, the MP and SCP of the Collembola are substantially lowered and in this way freezing is avoided. The increased cold hardiness by dehydration is similar to the protective dehydration mechanism described in earthworm cocoons and Arctic enchytraeids. Accepted: 5 January 1998     

Storage of Porcine Articular Cartilage at High Subzero Temperatures

Objective: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. Design: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (−10, −15, and −20 °C), and held for 30 min. After warming, 70 μm slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. Results: Results demonstrated excellent cell recovery (>75%) at −10°C provided ice did not form. Excellent cell recovery (>70%) occurred at −15°C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at −20°C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form Conclusions: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

Microbial communities in saline lakes of the Vestfold Hills (eastern Antarctica)

A detailed survey was undertaken of the microbial communities of 16 saline lakes in the Vestfold Hills (Princess Elizabeth Land, eastern Antarctica), which ranged in salinity from slightly brackish (4–5‰) to hypersaline (maximum: 174‰). Temperatures at comparable sampling depths in the lakes ranged from −12.2°C to +10.5°C. Ranges in the abundances of bacteria, heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were 1.40 × 10⁷ l⁻¹–1.58 × 10¹⁰ l⁻¹, 4.83 × 10⁴ l⁻¹–1.70 × 10⁷ l⁻¹ and 0–1.02 × 10⁷ l⁻¹, respectively. There was considerable variation across the salinity spectrum, though in the case of bacteria and PNAN significantly higher concentrations of cells were seen in two of the most saline lakes. The autotrophic ciliate Mesodinium rubrum occurred in all but five of the lakes and was found at salinity levels up to 108‰. Heterotrophic ciliates were generally scarce. Dinoflagellates, particularly Gonyaulax c.f. tamarensis, Gyrodinium lachryma and Gymnodinium sp., occurred in the majority of the lakes. On the basis of chlorophyll a concentrations, nutrient levels and microplankton concentrations the lakes spanned the spectrum from ultra-oligotrophic to oligo/mesotrophic. The most saline lakes had much reduced species diversity compared with the less saline environments. Isolation from the marine environment has led to nutrient depletion, simplification and a truncated trophic structure. Received: 19 September 1996 / Accepted: 13 January 1997     

Microorganism inactivation using high-pressure generation in sealed vessels under sub-zero temperature

In order to test the possibility of utilizing high pressure in bioscience and biotechnology, a simple method for high-pressure generation and its use for microbial inactivation have been studied. When a pressure vessel was filled with water, sealed tightly and cooled to sub-zero temperatures, high pressure was generated in the vessel. The pressure generation was 60 MPa at −5 °C, 103 MPa at −10 °C, and 140 MPa at −15 °C, −20 °C, and −22 °C. The high pressure generated inactivated microorganisms effectively: yeasts (Saccharomyces cerevisiae and Zygosaccharomyces rouxii), bacteria (Lactobacillus brevis and Eschericia coli), and fungi (Aspergillus niger and Aspergillus oryzae) were completely inactivated when stored in sealed vessels −20 °C for 24 h. However, Staphylococcus aureus was only partly inactivated under the same conditions. This method opens up a new application of high pressure for storing, transporting, and sterilizing of foods and biological materials. Received: 28 July 1997 / Received last revision: 12 June 1998 / Accepted: 19 June 1998     

Biodegradation of propanol and isopropanol by a mixed microbial consortium

The aerobic biodegradation of high concentrations of 1-propanol and 2-propanol (IPA) by a mixed microbial consortium was investigated. Solvent concentrations were one order of magnitude greater than any previously reported in the literature. The consortium utilized these solvents as their sole carbon source to a maximum cell density of 2.4 × 10⁹ cells ml⁻¹. Enrichment experiments with propanol or IPA as carbon sources were carried out in batch culture and maximum specific growth rates (μ_max) calculated. At 20 °C, μ _max values were calculated to be 0.0305 h⁻¹ and 0.1093 h⁻¹ on 1% (v/v) IPA and 1-propanol, respectively. Growth on propanol and IPA was carried out between temperatures of 10 °C and 45 °C. Temperature shock responses by the microbial consortium at temperatures above 45 °C were demonstrated by considerable cell flocculation. An increase in propanol substrate concentration from 1% (v/v) to 2% (v/v) decreased the μ _max from 0.1093 h⁻¹ to 0.0715 h⁻¹. Maximum achievable biodegradation rates of propanol and IPA were 6.11 × 10⁻³% (v/v) h⁻¹ and 2.72 × 10⁻³% (v/v) h⁻¹, respectively. Generation of acetone during IPA biodegradation commenced at 264 h and reached a maximum concentration of 0.4% (v/v). The results demonstrate the potential of mixed microbial consortia in the bioremediation of solvent-containing waste streams. Received: 14 December 1999 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Cold hardiness,adaptive strategies,and invasion of slugs of the genus <Emphasis Type="Italic">Deroceras</Emphasis> (Gastropoda,Pulmonata) in northeastern Asia

Abstract-Four species of slugs have been identified in the vicinity of Magadan: Deroceras laeve, D. altaicum, D. reticulatum, and D. agreste. They exemplify three different life cycle schemes, with D. reticulatum and D. altaicum wintering at the egg phase; D. laeve, at the slug phase; and D. agreste, at either phase. The D. altaicum and D. reticulatum slugs and D. laeve eggs are intolerant of subzero temperatures. D. laeve’s tolerate freezing and survive at temperatures below −28°C. The eggs of other species, which lose up to 35% of water upon cooling, can withstand temperatures as low as −15 to −17°C (D. altaicum), −25°C (D. agreste), and −35°C (D. reticulatum). According to preliminary data, D. agreste slugs survive at temperatures down to −10°C. The almost ubiquitous distribution of D. laeve in regions with cool summers (including zonal tundras) is accounted for not so much by the high rate of ontogeny as by its significant cold hardiness and ability to winter at different phases of the life cycle (except for the egg phase), which allows the period of development to be prolonged for the next seasons. The last is confirmed by the fact that the slugs collected before and after hibernation proved to have identical patterns of distribution by body weight. Three species of slugs introduced in the vicinity of Magadan fail to spread inland. In the case of D. reticulatum, this is explained primarily by the fact that the frost-free season in inland areas is too short to allow these slugs to complete ontogeny and lay eggs. The barriers to their expansion appear to be insuperable, since this process remains unsuccessful over no less than 75–80 years.     

Spring bloom succession,grazing impact and herbivore selectivity of ciliate communities in response to winter warming

This study aimed at simulating different degrees of winter warming and at assessing its potential effects on ciliate succession and grazing-related patterns. By using indoor mesocosms filled with unfiltered water from Kiel Bight, natural light and four different temperature regimes, phytoplankton spring blooms were induced and the thermal responses of ciliates were quantified. Two distinct ciliate assemblages, a pre-spring and a spring bloom assemblage, could be detected, while their formation was strongly temperature-dependent. Both assemblages were dominated by Strobilidiids; the pre-spring bloom phase was dominated by the small Strobilidiids Lohmaniella oviformis, and the spring bloom was mainly dominated by large Strobilidiids of the genus Strobilidium. The numerical response of ciliates to increasing food concentrations showed a strong acceleration by temperature. Grazing rates of ciliates and copepods were low during the pre-spring bloom period and high during the bloom ranging from 0.06 (Δ0°C) to 0.23 day⁻¹ (Δ4°C) for ciliates and 0.09 (Δ0°C) to 1.62 day⁻¹ (Δ4°C) for copepods. During the spring bloom ciliates and copepods showed a strong dietary overlap characterized by a wide food spectrum consisting mainly of Chrysochromulina sp., diatom chains and large, single-celled diatoms. Priority programme of the German Research Foundation—contribution 4.     

Effects of different ion compositions on growth of obligately halophilic protozoan Halocafeteria seosinensis

Substantial halophilic organisms have been found in 100–200‰ salinities. These ranges represent a highly specialized halophilic environment to which only a few halotolerant species have adapted. Recent studies have underlined the existence of diverse obligately halophilic protozoa in the salinity ranges of 100–200‰. The ranges of salinity under which these organisms can grow have been examined to some extent, but the balance of specific ions that will support growth has not been investigated. The heterotrophic nanoflagellate Halocafeteria, the type strain of which grows optimally at 150‰ salinity and 35°C, is a commonly encountered obligate halophile found in very hypersaline environments. These extreme environments can vary in their Mg:Ca ratios (i.e. weight ratios) and sulfate concentrations. To examine growth response of Halocafeteria to the different chemical compositions, densities of Halocafeteria seosinensis strain EHF34 were monitored in seven different ion composition media for 9 days at 1- to 2-day intervals (at 150‰ salinity and 35°C, with no prey limitation). Halocafeteria does not grow at Mg:Ca ratios of 35 and 100 and at high sulfate concentrations of 11.6 and 31.6 g l⁻¹. It grows well in 0.6 g l⁻¹ sulfate media at Mg:Ca ratios of 2, 10 or 35, but not 100. The present study demonstrates that the growth of the obligate halophile Halocafeteria can be affected by different ion compositions in hypersaline environments. Therefore, Halocafeteria may not be ubiquitous in hypersaline environments due to its ionic requirements.     

Cold-active halophilic bacteria from the ice-sealed Lake Vida,Antarctica

Lake Vida is a large, permanently ice-covered lake in the Victoria Valley of the McMurdo Dry Valleys, Antarctica and is unique among Dry Valley lakes because it is ice-sealed, with an ice-cover of nearly 19 m. Enrichment cultures of melt-water from Lake Vida 15.9 m ice yielded five pure cultures of aerobic, heterotrophic bacteria. Of these, one strain grew at −8°C and the four others at −4°C. All isolates were either halotolerant or halophilic, with two strains capable of growth at 15% NaCl. Phylogenetic analysis revealed the Lake Vida isolates to be Gammaproteobacteria, related to species of Psychrobacter and Marinobacter. This is the first report of pure cultures of bacteria from Lake Vida, and the isolates displayed a phenotype consistent with life in a cold hypersaline environment.     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

Cold-induced changes of enzymes,proline, carbohydrates,and chlorophyll in wheat

Quantitative changes in total leaf soluble proteins, proline, carbohydrate content, chlorophyll fluorescence, guaiacol peroxidase (POD) and catalase (CAT) activities were determined in a less cold-hardy (LCH) spring cv. Kohdasht (LT₅₀ = −6°C), a semi cold-hardy (SCH) facultative cv. Azar 2 (LT₅₀ = −15°C), and a cold-hardy (CH) winter cv. Norstar (LT₅₀ = −26°C) of wheat (Triticum aestivum L.) exposed to 4°C for 9 weeks. Seedlings were grown in a controlled growth room for 14 days at 20°C and then transferred to 4°C (experimental day 0) for 63 days (cold treatment); otherwise they were maintained continuously at 20°C (control treatment). The samples were harvested 0, 2, 21, 28, 42, and 63 days after exposure to 4°C. The results showed significant low temperature (LT)-induced accumulation of total soluble proteins, proline, and carbohydrates and elevation in activities of CAT and POD in leaves of SCH and CH winter cultivars rather than in LCH spring cultivar. In contrast, the chlorophyll fluorescence (F _v/F _m) declined during LT treatment irrespective of cultivar. The results suggest that developmental traits such as vernalization requirement of wheat affects on cold-tolerance expression system of plants.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

Microbial sequences retrieved from environmental samples from seasonal Arctic snow and meltwater from Svalbard, Norway

16S rRNA gene (rrs) clone libraries were constructed from two snow samples (May 11, 2007 and June 7, 2007) and two meltwater samples collected during the spring of 2007 in Svalbard, Norway (79°N). The libraries covered 19 different microbial classes, including Betaproteobacteria (21.3%), Sphingobacteria (16.4%), Flavobacteria (9.0%), Acidobacteria (7.7%) and Alphaproteobacteria (6.5%). Significant differences were detected between the two sets of sample libraries. First, the meltwater libraries had the highest community richness (Chao1: 103.2 and 152.2) and Shannon biodiversity indices (between 3.38 and 3.59), when compared with the snow libraries (Chao1: 14.8 and 59.7; Shannon index: 1.93 and 3.01). Second, ∫-LIBSHUFF analyses determined that the bacterial communities in the snow libraries were significantly different from those of the meltwater libraries. Despite these differences, our data also support the theory that a common core group of microbial populations exist within a variety of cryohabitats.     

Characterization of a recombinant (?)γ-lactamase from Microbacterium hydrocarbonoxydans

A (−)γ-lactamase, Mhg, from Microbacterium hydrocarbonoxydans was over-expressed in E. coli and was characterized after purification. The maximum activity was at pH 8.0 and 60°C and the half life of Mhg was ~30 min at 75°C. The enzyme was activated by DTT. The catalytic triad of the (−)γ-lactamase is comprised of residues Ser98, Asp230, and His259 and an oxyanion hole was formed by Tyr32 and Met99 according to the alignment results. Under native conditions, the (−)γ-lactamase consists of two 31 kDa homodimers.     

Bacterial Succession within an Ephemeral Hypereutrophic Mojave Desert Playa Lake

Ephemerally wet playas are conspicuous features of arid landscapes worldwide; however, they have not been well studied as habitats for microorganisms. We tracked the geochemistry and microbial community in Silver Lake playa, California, over one flooding/desiccation cycle following the unusually wet winter of 2004–2005. Over the course of the study, total dissolved solids increased by ∽10-fold and pH increased by nearly one unit. As the lake contracted and temperatures increased over the summer, a moderately dense planktonic population of ∽1 × 10⁶ cells ml⁻¹ of culturable heterotrophs was replaced by a dense population of more than 1 × 10⁹ cells ml⁻¹, which appears to be the highest concentration of culturable planktonic heterotrophs reported in any natural aquatic ecosystem. This correlated with a dramatic depletion of nitrate as well as changes in the microbial community, as assessed by small subunit ribosomal RNA gene sequencing of bacterial isolates and uncultivated clones. Isolates from the early-phase flooded playa were primarily Actinobacteria, Firmicutes, and Bacteroidetes, yet clone libraries were dominated by Betaproteobacteria and yet uncultivated Actinobacteria. Isolates from the late-flooded phase ecosystem were predominantly Proteobacteria, particularly alkalitolerant isolates of Rhodobaca, Porphyrobacter, Hydrogenophaga, Alishwenella, and relatives of Thauera; however, clone libraries were composed almost entirely of Synechococcus (Cyanobacteria). A sample taken after the playa surface was completely desiccated contained diverse culturable Actinobacteria typically isolated from soils. In total, 205 isolates and 166 clones represented 82 and 44 species-level groups, respectively, including a wide diversity of Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Gemmatimonadetes, Acidobacteria, and Cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.