Closely related strains of thermophilic Synechococcus were cultivated from the microbial mats found in the effluent channels of Mushroom Spring, Yellowstone National Park (YNP). These strains have identical or nearly identical 16S rRNA sequences but are representative of separate, predicted putative ecotype (PE) populations, which were identified by using the more highly resolving psaA locus and which predominate at different vertical positions within the 1-mm-thick upper-green layer of the mat. Pyrosequencing confirmed that each strain contained a single, predominant psaA genotype. Strains differed in growth rate as a function of irradiance. A strain with a psaA genotype corresponding to a predicted PE that predominates near the mat surface grew fastest at high irradiances, whereas strains with psaA genotypes representative of predominant subsurface populations grew faster at low irradiance and exhibited greater sensitivity to abrupt shifts to high light. The high-light-adapted and low-light-adapted strains also exhibited differences in pigment content and the composition of the photosynthetic apparatus (photosystem ratio) when grown under different light intensities. Cells representative of the different strains had similar morphologies under low-light conditions, but under high-light conditions, cells of low-light-adapted strains became elongated and formed short chains of cells. Collectively, the results presented here are consistent with the hypothesis that closely related, but distinct, ecological species of Synechococcus occupy different light niches in the Mushroom Spring microbial mat and acclimate differently to changing light environments.

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level.

The construction and testing of a unique instrument, the Paleobiosphere, which mimics some of the conditions of the ancient earth, is described. The instrument provides an experimental testing system for determining if certain microbes, when provided an adequate environment, can degrade biological materials to produce fuel-like hydrocarbons in a relatively short time frame that become trapped by the shale. The conditions selected for testing included a particulate Montana shale (serving as the "Trap Shale"), plant materials (leaves and stems of three extant species whose origins are in the late Cretaceous), a water-circulating system, sterile air, and a specially designed Carbotrap through which all air was passed as exhaust and volatile were hydrocarbons trapped. The fungus for initial testing was Annulohypoxylon sp., isolated as an endophyte of Citrus aurantifolia. It produces, in solid and liquid media, a series of hydrocarbon-like molecules. Some of these including 1,8-cineole, 2-butanone, propanoic acid, 2-methyl-, methyl ester, benzene (1-methylethyl)-, phenylethyl alcohol, benzophenone and azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl), [1S-(1α,7α,8aβ)]. These were the key signature compounds used in an initial Paleobiosphere test. After 3 weeks, incubation, the volatiles associated with the harvested "Trap Shale" included each of the signature substances as well as other fungal-associated products: some indanes, benzene derivatives, some cyclohexanes, 3-octanone, naphthalenes and others. The fungus thus produced a series of "Trap Shale" products that were representative of each of the major classes of hydrocarbons in diesel fuel (Mycodiesel). Initial tests with the Paleobiosphere offer some evidence for a possible origin of hydrocarbons trapped in bentonite shale. Thus, with modifications, numerous other tests can also be designed for utilization in the Paleobiosphere.

Gloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated "synergistans." An expression of the "median synergistic effect," under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50% inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed.

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single cell level.