Blake W. Stamps

Metabolic Capability of a Predominant Halanaerobium sp. in Hydraulically Fractured Gas Wells and Its Implication in Pipeline Corrosion

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O32-, SO42-, and HS-). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure.

Hoeflea anabaenae sp. nov., an epiphytic symbiont that attaches to the heterocysts of a strain of Anabaena

The heterotrophic, epiphytic, symbiotic bacterial strain WH2K(T) was previously isolated from a two-member culture in which it was attached to the heterocysts of a strain of Anabaena (SSM-00). Analysis of its 16S rRNA gene sequence demonstrated that the symbiont was most closely related to the type strain of Hoeflea marina (96.9 % similarity), which belongs to the family Phyllobacteriaceae within the order Rhizobiales of the class Alphaproteobacteria. A polyphasic taxonomic study was performed on strain WH2K(T), which consisted of irregular rods (2-5 µm long, 0.2 µm wide) that appeared to be narrower at one pole. Optimal growth was obtained in complex media with 15 g sea salts l(-1), at 18-34 °C (30 °C optimum) and at pH 6.0-8.0 (optimum pH 6.5). Unknown growth requirements were provided by small amounts of yeast extract but not by standard vitamin and trace metal solutions. Of the substrates tested, WH2K(T) was able to utilize only acetate, pyruvate, malate and fumarate. Growth was observed only under aerobic and microaerobic conditions, and nitrate was not reduced. No photosynthetic pigments were detected under any of the growth conditions tested. The predominant fatty acids were a summed feature that comprises C(18 : 1)ω7c, C(18 : 1)ω9t, C(18 : 1)ω12t or any combination of these (64.0 %) and an unidentified fatty acid of equivalent chain length 17.603 (13.5 %). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphoglycolipid, unknown lipids and an unidentified aminolipid. The only respiratory ubiquinone detected was Q-10. The DNA G+C content of the strain was 58.1 mol%. The organism can form a site-specific attached symbiotic relationship with a species of Anabaena. Based on phylogenetic and phenotypic evidence, it is proposed that strain WH2K(T) be classified within a novel species of the genus Hoeflea, for which the name Hoeflea anabaenae sp. nov. is proposed. The type strain is WH2K(T) ( = CCUG 56626(T)  = NRRL B-59520(T)).

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

Municipal Solid Waste Landfills Harbor Distinct Microbiomes

Landfills are the final repository for most of the discarded material from human society and its “built environments.” Microorganisms subsequently degrade this discarded material in the landfill, releasing gases (largely CH4 and CO2) and a complex mixture of soluble chemical compounds in leachate. Characterization of “landfill microbiomes” and their comparison across several landfills should allow the identification of environmental or operational properties that influence the composition of these microbiomes and potentially their biodegradation capabilities. To this end, the composition of landfill microbiomes was characterized as part of an ongoing USGS national survey studying the chemical composition of leachates from 19 non-hazardous landfills across 16 states in the continental U.S. The landfills varied in parameters such as size, waste composition, management strategy, geography, and climate zone. The diversity and composition of bacterial and archaeal populations in leachate samples were characterized by 16S rRNA gene sequence analysis, and compared against a variety of physical and chemical parameters in an attempt to identify their impact on selection. Members of the Epsilonproteobacteria, Gammaproteobacteria, Clostridia, and candidate division OP3 were the most abundant. The distribution of the observed phylogenetic diversity could best be explained by a combination of variables and was correlated most strongly with the concentrations of chloride and barium, rate of evapotranspiration, age of waste, and the number of detected household chemicals. This study illustrates how leachate microbiomes are distinct from those of other natural or built environments, and sheds light on the major selective forces responsible for this microbial diversity.

Draft Genome of a Novel Chlorobi Member Assembled by Tetranucleotide Binning of a Hot Spring Metagenome

ABSTRACT The genome of a member of the phylum Chlorobi was assembled from a shotgun metagenomic sequence of a hot spring in Mammoth Lakes, CA. This organism appears to be a novel, aerobic, photosynthetic Chlorobi member, expanding the knowledge of this underrepresented phylum.

Genomic and Metabolomic Insights into the Natural Product Biosynthetic Diversity of a Feral-Hog-Associated Brevibacillus laterosporus Strain

Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolates secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine (L-phenylalanol). An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals.

The deep, hot biosphere: Twenty-five years of retrospection

Twenty-five years ago this month, Thomas Gold published a seminal manuscript suggesting the presence of a “deep, hot biosphere” in the Earth’s crust. Since this publication, a considerable amount of attention has been given to the study of deep biospheres, their role in geochemical cycles, and their potential to inform on the origin of life and its potential outside of Earth. Overwhelming evidence now supports the presence of a deep biosphere ubiquitously distributed on Earth in both terrestrial and marine settings. Furthermore, it has become apparent that much of this life is dependent on lithogenically sourced high-energy compounds to sustain productivity. A vast diversity of uncultivated microorganisms has been detected in subsurface environments, and we show that H2, CH4, and CO feature prominently in many of their predicted metabolisms. Despite 25 years of intense study, key questions remain on life in the deep subsurface, including whether it is endemic and the extent of its involvement in the anaerobic formation and degradation of hydrocarbons. Emergent data from cultivation and next-generation sequencing approaches continue to provide promising new hints to answer these questions. As Gold suggested, and as has become increasingly evident, to better understand the subsurface is critical to further understanding the Earth, life, the evolution of life, and the potential for life elsewhere. To this end, we suggest the need to develop a robust network of interdisciplinary scientists and accessible field sites for long-term monitoring of the Earth’s subsurface in the form of a deep subsurface microbiome initiative.

Genome Sequence of Thermoanaerobaculum aquaticum MP-01T, the First Cultivated Member of Acidobacteria Subdivision 23, Isolated from a Hot Spring.

ABSTRACT Thermoanaerobaculum aquaticum MP-01T is currently the only cultivated and described member of Acidobacteria subdivision 23. Here, we report the genome sequence for this novel microorganism that was isolated from a hot spring.

Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.

Carbonate-rich dendrolitic cones: insights into a modern analog for incipient microbialite formation, Little Hot Creek, Long Valley Caldera, California

Ancient putative microbial structures that appear in the rock record commonly serve as evidence of early life on Earth, but the details of their formation remain unclear. The study of modern microbial mat structures can help inform the properties of their ancient counterparts, but modern mineralizing mat systems with morphological similarity to ancient structures are rare. Here, we characterize partially lithified microbial mats containing cm-scale dendrolitic coniform structures from a geothermal pool (“Cone Pool”) at Little Hot Creek, California, that if fully lithified, would resemble ancient dendrolitic structures known from the rock record. Light and electron microscopy revealed that the cm-scale ‘dendrolitic cones’ were comprised of intertwined microbial filaments and grains of calcium carbonate. The degree of mineralization (carbonate content) increased with depth in the dendrolitic cones. Sequencing of 16S rRNA gene libraries revealed that the dendrolitic cone tips were enriched in OTUs most closely related to the genera Phormidium, Leptolyngbya, and Leptospira, whereas mats at the base and adjacent to the dendrolitic cones were enriched in Synechococcus. We hypothesize that the consumption of nutrients during autotrophic and heterotrophic growth may promote movement of microbes along diffusive nutrient gradients, and thus microbialite growth. Hour-glass shaped filamentous structures present in the dendrolitic cones may have formed around photosynthetically-produced oxygen bubbles—suggesting that mineralization occurs rapidly and on timescales of the lifetime of a bubble. The dendrolitic-conical structures in Cone Pool constitute a modern analog of incipient microbialite formation by filamentous microbiota that are morphologically distinct from any structure described previously. Thus, we provide a new model system to address how microbial mats may be preserved over geological timescales.Paleobiology: modern microbes may tell an ancient taleMicrobial mats currently thriving in a hot pool in California may help explain the origin of fossilized evidence of early life on Earth. Modern microbial mats that are structurally similar to microbial fossil mats are rare. John Spear at the Colorado School of Mines, with co-workers from elsewhere in the USA and in Chile, examined the microbial mats growing in a geothermal pool at Little Hot Creek in California. Light microscopy and electron microscopy identified crucial fine structure similarities with branching mat structures in the fossil record. The researchers developed hypotheses to explain the influence of nutrient flow on the growth and movement of the microbes in the mats. These living mats are a useful model system to help researchers understand how ancient microbial mats formed and were preserved over geological timescales.