a-set-situ-informed-simulated-medium-formats-for-culturing

A Set of <em>In Situ</em> Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Southern Illinois University Carbondale

Chewed and expectorated quids, indigestible stringy fibers from the roasted inner pulp of agave or yucca root, have proven resilient over long periods of time in dry cave environments and correspondingly, although little studied, are common in archaeological archives. In the late 1960s, thousands of quids were recovered from Mule Spring Rockshelter (Nevada, USA) deposits and stored without consideration to DNA preservation in a museum collection, remaining unstudied for over fifty years. To assess the utility of these materials as repositories for genetic information about past inhabitants of the region and their movements, twenty-one quids were selected from arbitrary excavation depths for detailed analysis. Human mitochondrial DNA sequences from the quids were amplified by PCR and screened for diagnostic single nucleotide polymorphisms. Most detected single nucleotide polymorphisms were consistent with recognized Native American haplogroup subclades B2a5, B2i1, C1, C1c, C1c2, and D1; with the majority of the sample set consistent with subclades C1, C1c, and C1c2. In parallel with the DNA analysis, each quid was radiocarbon dated, revealing a time-resolved pattern of occupancy from 347 to 977 calibrated years before present. In particular, this dataset reveals strong evidence for the presence of haplogroup C1/C1c at the Southwestern edge of the US Great Basin from ~670 to 980 cal YBP, which may temporally correspond with the beginnings of the so-called Numic Spread into the region. The research described here demonstrates an approach which combines targeted DNA analysis with radiocarbon age dating; thus enabling the genetic analysis of archaeological materials of uncertain stratigraphic context. Here we present a survey of the maternal genetic profiles from people who used the Mule Spring Rockshelter and the historic timing of their utilization of a key natural resource.

Ancient human mitochondrial DNA and radiocarbon analysis of archived quids from the Mule Spring Rockshelter, Nevada, USA.

Deep fractured rock ecosystems across most of North America have not been studied extensively. However, the US Great Basin, in particular the Nevada National Security Site (NNSS, formerly the Nevada Test Site), has hosted a number of influential subsurface investigations over the years. This investigation focuses on resident microbiota recovered from a hydrogeologically confined aquifer in fractured Paleozoic carbonate rocks at 863 - 923 meters below land surface. Analysis of the microorganisms living in this oligotrophic environment provides a perspective into microbial metabolic strategies required to endure prolonged hydrogeological isolation deep underground. Here we present a microbiological and physicochemical characterization of a deep continental carbonate ecosystem and describe a bacterial genus isolated from the ecosystem. Strain DRI-13 is a strictly anaerobic, moderately thermophilic, fumarate-respiring member of the phylum . This bacterium grows optimally at 55°C and pH 8.0, can tolerate a concentration of 100 mM NaCl, and appears to obligately metabolize fumarate to acetate and succinate. Culture-independent 16S rRNA gene sequencing indicates a global subsurface distribution, while the closest cultured relatives of DRI-13 are  (90.0% similarity) and  (88.0% similarity). The predominant fatty acid profile is iso-C  , C  , C   and C  . The percentage of the straight-chain fatty acid C   is a defining characteristic not present in the other closely related species. The genome is estimated to be 3,649,665 bp, composed of 87.3% coding regions with an overall average of 45.1% G + C content. Strain DRI-13 represents a novel genus of subsurface bacterium isolated from a previously uncharacterized rock-hosted geothermal habitat. The characterization of the bacterium combined with the sequenced genome provides insights into metabolism strategies of the deep subsurface biosphere. Based on our characterization analysis we propose the name  (DRI-13 = DSM 100382 = ATCC TSD-12).

 gen. nov. sp. nov., a Novel Fumarate-Fermenting Microorganism From a Deep Fractured Carbonate Aquifer of the US Great Basin.

 (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO, and H) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO as a unique source of carbon with increased solvent formation and atmospheric CO reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO served as the only source of carbon. In the current study, 55 mutagenized  mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55  mutants showed a significant increase in ethanol, butyrate, and butanol production. The three  mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3  mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO only). In addition, one  mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO conditions. Most of the  mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the  mutant presented 0.082 g/Abs of CO reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO reduction efficiency.

EMS-Induced Mutagenesis of  for Increased Atmospheric CO Reduction Efficiency and Solvent Production.

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of  experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two  OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant  and  sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as , , and . Subsequent experimental deployments included a methanogen-dominated stage ( and ) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of  time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.

Patterns of  Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer.

There is a dearth of sequenced and closed microbial genomes from environments that exceed > 500 m below level terrestrial surface. Coupled with even fewer cultured isolates, study and understanding of how life endures in the extreme oligotrophic subsurface environments is greatly hindered. Using a de novo hybrid assembly of Illumina and Oxford Nanopore sequences we produced a circular genome with corresponding methylome profile of the recently characterized thermophilic, anaerobic, and fumarate-respiring subsurface bacterium, Thermanaerosceptrum fracticalcis, strain DRI-13 to understand how this microorganism survives the deep subsurface.

Hybrid genome de novo assembly with methylome analysis of the anaerobic thermophilic subsurface bacterium Thermanaerosceptrum fracticalcis strain DRI-13.

The Hawaiian Bobtail Squid (Euprymna scolopes) is a model organism for investigating host-symbiont relationships. The current scientific focus is on the microbiome within E. scolopes, while very little is known about the microbiome of the tanks housing E. scolopes. We examined the hypothesis that bacterial communities and geochemistry within the squid tank environment correlate with the production of viable paralarval squid.

Aquaculture production of hatchling Hawaiian Bobtail Squid (Euprymna scolopes) is negatively impacted by decreasing environmental microbiome diversity.

Subsurface microbial community distribution patterns are influenced by biogeochemical and groundwater fluxes and may inform hydraulic connections along groundwater-flow paths. This study examined the regional-scale microbial community of the Death Valley Regional Flow System and evaluated whether subsurface communities can be used to identify groundwater-flow paths between recharge and discharge areas. Samples were collected from 36 sites in three groundwater basins: Pahute Mesa-Oasis Valley (PMOV), Ash Meadows (AM), and Alkali Flat-Furnace Creek Ranch (AFFCR). Microbial diversity within and between communities varied by location, and communities were separated into two overall groups that affiliated with the AM and PMOV/AFFCR basins. Network analysis revealed patterns between clusters of common microbes that represented groundwaters with similar geochemical conditions and largely corroborated hydraulic connections between recharge and discharge areas. Null model analyses identified deterministic and stochastic ecological processes contributing to microbial community assemblages. Most communities were more different than expected and governed by dispersal limitation, geochemical differences, or undominating processes. However, certain communities from sites located within or near the Nevada National Security Site were more similar than expected and dominated by homogeneous dispersal or selection. Overall, the (dis)similarities between the microbial communities of DVRFS recharge and discharge areas supported previously documented hydraulic connections between: (1) Spring Mountains and Ash Meadows; (2) Frenchman and Yucca Flat and Amargosa Desert; and (3) Amargosa Desert and Death Valley. However, only a portion of the flow path between Pahute Mesa and Oasis Valley could be supported by microbial community analyses, likely due to well-associated artifacts in samples from the two Oasis Valley sites. This study demonstrates the utility of combining microbial data with hydrologic, geologic, and water-chemistry information to comprehensively characterize groundwater systems, highlighting both strengths and limitations of this approach.

Article	Year
Ancient human mitochondrial DNA and radiocarbon analysis of archived quids from the Mule Spring Rockshelter, Nevada, USA. PloS one\| PubMed ID: 29522562	2018
gen. nov. sp. nov., a Novel Fumarate-Fermenting Microorganism From a Deep Fractured Carbonate Aquifer of the US Great Basin. Frontiers in microbiology\| PubMed ID: 31611860	2019
EMS-Induced Mutagenesis of for Increased Atmospheric CO Reduction Efficiency and Solvent Production. Microorganisms\| PubMed ID: 32824093	2020
Patterns of Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer. Frontiers in microbiology\| PubMed ID: 33329414	2020
Hybrid genome de novo assembly with methylome analysis of the anaerobic thermophilic subsurface bacterium Thermanaerosceptrum fracticalcis strain DRI-13. BMC genomics\| PubMed ID: 33757423	2021
Aquaculture production of hatchling Hawaiian Bobtail Squid (Euprymna scolopes) is negatively impacted by decreasing environmental microbiome diversity. Journal of applied microbiology\| PubMed ID: 34724303	2022
Subsurface microbial communities as a tool for characterizing regional-scale groundwater flow. The Science of the total environment\| PubMed ID: 35738377	2022

Scott D. Hamilton-Brehm

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