In situ Linkage of Fungal and Bacterial Proliferation to Microbiologically Influenced Corrosion in B20 Biodiesel Storage Tanks.

Renewable fuels hold great promise for the future yet their susceptibility to biodegradation and subsequent corrosion represents a challenge that needs to be directly assessed. Biodiesel is a renewable fuel that is widely used as a substitute or extender for petroleum diesel and is composed of a mixture of fatty acid methyl esters derived from plant or animal fats. Biodiesel can be blended up to 20% v/v with ultra-low sulfur diesel (i.e., B20) and used interchangeably with diesel engines and infrastructure. The addition of biodiesel, however, has been linked to increased susceptibility to biodegradation. Microorganisms proliferating via degradation of biodiesel blends have been linked to microbiologically influenced corrosion in the laboratory, but not measured directly in storage tanks (i.e., in situ). To measure in situ microbial proliferation, fuel degradation and microbially influenced corrosion, we conducted a yearlong study of B20 storage tanks in operation at two locations, identified the microorganisms associated with fuel fouling, and measured in situ corrosion. The bacterial populations were more diverse than the fungal populations, and largely unique to each location. The bacterial populations included members of the Acetobacteraceae, Clostridiaceae, and Proteobacteria. The abundant Eukaryotes at both locations consisted of the same taxa, including a filamentous fungus within the family Trichocomaceae, not yet widely recognized as a contaminant of petroleum fuels, and the Saccharomycetaceae family of yeasts. Increases in the absolute and relative abundances of the Trichocomaceae were correlated with significant, visible fouling and pitting corrosion. This study identified the relationship between fouling of B20 with increased rates of corrosion and the microorganisms responsible, largely at the bottom of the sampled storage tanks. To our knowledge this is the first in situ study of this scale incorporating community and corrosion measurements in an active biodiesel storage environment.

Draft Genome Sequence of Filamentous Fungus Phialemoniopsis curvata, Isolated from Diesel Fuel.

Phialemoniopsis curvata D216 is a filamentous fungus isolated from contaminated diesel fuel. The genome size is 40.3 Mbp with a G+C content of 54.81%. Its genome encodes enzymes and pathways likely involved in the degradation of and survival in fuel, including lipases, fatty acid transporters, and beta oxidation.

Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML.

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics and potential metabolic function during an intense natural algal bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate-reducing microorganisms throughout the lake's water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sediment to repopulate the lake water column as the bloom subsides.IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to understand how the microbial community responds to human-influenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community responds during a bloom at Mono Lake.

Environmental and Biological Influences on Carbonate Precipitation Within Hot Spring Microbial Mats in Little Hot Creek, CA.

Microbial mats are found in a variety of modern environments, with evidence for their presence as old as the Archean. There is much debate about the rates and conditions of processes that eventually lithify and preserve mats as microbialites. Here, we apply novel tracer experiments to quantify both mat biomass addition and the formation of CaCO3. Microbial mats from Little Hot Creek (LHC), California, contain calcium carbonate that formed within multiple mat layers, and thus constitute a good test case to investigate the relationship between the rate of microbial mat growth and carbonate precipitation. The laminated LHC mats were divided into four layers via color and fabric, and waters within and above the mat were collected to determine their carbonate saturation states. Samples of the microbial mat were also collected for 16S rRNA analysis of microbial communities in each layer. Rates of carbonate precipitation and carbon fixation were measured in the laboratory by incubating homogenized samples from each mat layer with δ13C-labeled HCO3- for 24 h. Comparing these rates with those from experimental controls, poisoned with NaN3 and HgCl2, allowed for differences in biogenic and abiogenic precipitation to be determined. Carbon fixation rates were highest in the top layer of the mat (0.17% new organic carbon/day), which also contained the most phototrophs. Isotope-labeled carbonate was precipitated in all four layers of living and poisoned mat samples. In the top layer, the precipitation rate in living mat samples was negligible although abiotic precipitation occurred. In contrast, the bottom three layers exhibited biologically enhanced carbonate precipitation. The lack of correlation between rates of carbon fixation and biogenic carbonate precipitation suggests that processes other than autotrophy may play more significant roles in the preservation of mats as microbialites.

Microscale Biosignatures and Abiotic Mineral Authigenesis in Little Hot Creek, California.

Hot spring environments can create physical and chemical gradients favorable for unique microbial life. They can also include authigenic mineral precipitates that may preserve signs of biological activity on Earth and possibly other planets. The abiogenic or biogenic origins of such precipitates can be difficult to discern, therefore a better understanding of mineral formation processes is critical for the accurate interpretation of biosignatures from hot springs. Little Hot Creek (LHC) is a hot spring complex located in the Long Valley Caldera, California, that contains mineral precipitates composed of a carbonate base (largely submerged) topped by amorphous silica (largely emergent). The precipitates occur in close association with microbial mats and biofilms. Geological, geochemical, and microbiological data are consistent with mineral formation via degassing and evaporation rather than direct microbial involvement. However, the microfabric of the silica portion is stromatolitic in nature (i.e., wavy and finely laminated), suggesting that abiogenic mineralization has the potential to preserve textural biosignatures. Although geochemical and petrographic evidence suggests the calcite base was precipitated via abiogenic processes, endolithic microbial communities modified the structure of the calcite crystals, producing a textural biosignature. Our results reveal that even when mineral precipitation is largely abiogenic, the potential to preserve biosignatures in hot spring settings is high. The features found in the LHC structures may provide insight into the biogenicity of ancient Earth and extraterrestrial rocks.

Genome Sequence of a Byssochlamys sp. Strain Isolated from Fouled B20 Biodiesel.

Byssochlamys sp. strain AF001 is a filamentous fungus isolated from fouled B20 biodiesel. Its growth on B20 biodiesel results in the degradation and fouling of the fuel and higher rates of corrosion in affected storage tanks. The genome of Byssochlamys sp. AF001 is 35.9 Mbp and is composed of 10 scaffolds, with a G+C content of 45.89%.

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.

Design features of offshore oil production platforms influence their susceptibility to biocorrosion.

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of 35S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (<37 °C) sites in all platforms had more SRB and higher SRA compared to samples from sites with higher temperatures and flow rates. However, high (5 mmol L-1) levels of hydrogen sulfide and high numbers (107 mL-1) of SRB were found in only one platform. This platform alone contained large separation tanks with long retention times and recycled fluids from stagnant sites to the beginning of the oil separation train, thus promoting distribution of biocorrosive microorganisms. These findings tell us that tracking microbial sulfate-reducing activity and community composition on off-shore oil production platforms can be used to identify operational practices that inadvertently promote the proliferation, distribution, and activity of biocorrosive microorganisms.

Local and Regional Diversity Reveals Dispersal Limitation and Drift as Drivers for Groundwater Bacterial Communities from a Fractured Granite Formation.

Microorganisms found in terrestrial subsurface environments make up a large proportion of the Earth's biomass. Biogeochemical cycles catalyzed by subsurface microbes have the potential to influence the speciation and transport of radionuclides managed in geological repositories. To gain insight on factors that constrain microbial processes within a formation with restricted groundwater flow we performed a meta-community analysis on groundwater collected from multiple discrete fractures underlying the Chalk River Laboratories site (located in Ontario, Canada). Bacterial taxa were numerically dominant in the groundwater. Although these were mainly uncultured, the closest cultivated representatives were from the phenotypically diverse Betaproteobacteria, Deltaproteobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, and Firmicutes. Hundreds of taxa were identified but only a few were found in abundance (>1%) across all assemblages. The remainder of the taxa were low abundance. Within an ecological framework of selection, dispersal and drift, the local and regional diversity revealed fewer taxa within each assemblage relative to the meta-community, but the taxa that were present were more related than predicted by chance. The combination of dispersion at one phylogenetic depth and clustering at another phylogenetic depth suggest both niche (dispersion) and filtering (clustering) as drivers of local assembly. Distance decay of similarity reveals apparent biogeography of 1.5 km. Beta diversity revealed greater influence of selection at shallow sampling locations while the influences of dispersal limitation and randomness were greater at deeper sampling locations. Although selection has shaped each assemblage, the spatial scale of groundwater sampling favored detection of neutral processes over selective processes. Dispersal limitation between assemblages combined with local selection means the meta-community is subject to drift, and therefore, likely reflects the differential historical events that have influenced the current bacterial composition. Categorizing the study site into smaller regions of interest of more closely spaced fractures, or of potentially hydraulically connected fractures, might improve the resolution of an analysis to reveal environmental influences that have shaped these bacterial communities.

Extractables analysis of single-use flexible plastic biocontainers.

UNLABELLED: Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.

A cytotoxic leachable compound from single-use bioprocess equipment that causes poor cell growth performance.

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid-handling units with disposable plastic bags. Such single-use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell-growth-based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held-media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components.

Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers.

Out of the plethora of chemical species extractable at low levels from the materials of construction of single-use bioprocess containers, we have identified one particularly conspicuous compound and shown it to be highly detrimental to cell growth. The compound, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), is derived from the breakdown of tris(2,4-di-tert-butylphenyl)phosphite (trade name Irgafos 168®), a common antioxidant additive present in many formulations of polyethylene (one of the polymers commonly used as the material contacting process fluids in bioprocess containers). Cell growth experiments using several mammalian cell lines and growth media spiked with bDtBPP show harmful effects at concentrations well below the parts-per-million range. Cellular response to bDtBPP is rapid, and results in a significant decrease in mitochondrial membrane potential. The migration of bDtBPP from polyethylene-based films is shown to be time- and temperature-dependent. Further, experiments suggest that exposure of oxidized Irgafos 168 to ionizing radiation (such as gamma irradiation) is an important condition for the generation of significant amounts of leachable bDtBPP. LAY ABSTRACT: Biopharmaceuticals are drugs manufactured using cells that are genetically engineered to produce a therapeutic protein. A current trend in biomanufacturing is the replacement of hard-plumbed stainless steel vessels (where these cells are grown) with specialized, pre-sterilized, disposable plastic bags. While this move has significant environmental and cost benefits, the effect of plastics on the biomanufacturing process is not yet completely understood. Here we show that if a chemical compound formed by the breakdown of a common antioxidant additive to plastics leaches into the cell culture liquid, the growth of mammalian cells is strongly inhibited. Some of the factors that promote the generation of this compound, and the conditions that favor migration of the compound into process fluids, are explored here.