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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A miniaturized feedstocks-to-fuels pipeline for screening the efficiency of deconstruction and microbial conversion of lignocellulosic biomass

Sustainably grown biomass is a promising alternative to produce fuels and chemicals and reduce the dependency on fossil energy sources. However, the efficient conversion of lignocellulosic biomass into biofuels and bioproducts often requires extensive testing of components and reaction conditions used in the pretreatment, saccharification, and bioconversion steps. This restriction can result in a significant and unwieldy number of combinations of biomass types, solvents, microbial strains, and operational parameters that need to be characterized, turning these efforts into a daunting and time-consuming task. Here we developed a high-throughput feedstocks-to-fuels screening platform to address these challenges. The result is a miniaturized semi-automated platform that leverages the capabilities of a solid handling robot, a liquid handling robot, analytical instruments, and a centralized data repository, adapted to operate as an ionic-liquid-based biomass conversion pipeline. The pipeline was tested by using sorghum as feedstock, the biocompatible ionic liquid cholinium phosphate as pretreatment solvent, a “one-pot” process configuration that does not require ionic liquid removal after pretreatment, and an engineered strain of the yeast Rhodosporidium toruloides that produces the jet-fuel precursor bisabolene as a conversion microbe. By the simultaneous processing of 48 samples, we show that this configuration and reaction conditions result in sugar yields (~70%) and bisabolene titers (~1500 mg/L) that are comparable to the efficiencies observed at larger scales but require only a fraction of the time. We expect that this Feedstocks-to-Fuels pipeline will become an effective tool to screen thousands of bioenergy crop and feedstock samples and assist process optimization efforts and the development of predictive deconstruction approaches.

09 BIOMASS FUELS↗
DOE OSTI2021

An Integrative Model for Soil Biogeochemistry and Methane Processes: I. Model Structure and Sensitivity Analysis

Abstract Environmental changes are anticipated to generate substantial impacts on carbon cycling in peatlands, affecting terrestrial‐climate feedbacks. Understanding how peatland methane (CH 4 ) fluxes respond to these changing environments is critical for predicting the magnitude of feedbacks from peatlands to global climate change. To improve predictions of CH 4 fluxes in response to changes such as elevated atmospheric CO 2 concentrations and warming, it is essential for Earth system models to include increased realism to simulate CH 4 processes in a more mechanistic way. To address this need, we incorporated a new microbial‐functional group‐based CH 4 module into the Energy Exascale Earth System land model (ELM) and tested it with multiple observational data sets at an ombrotrophic peatland bog in northern Minnesota. The model is able to simulate observed land surface CH 4 fluxes and fundamental mechanisms contributing to these throughout the soil profile. The model reproduced the observed vertical distributions of dissolved organic carbon and acetate concentrations. The seasonality of acetoclastic and hydrogenotrophic methanogenesis—two key processes for CH 4 production—and CH 4 concentration along the soil profile were accurately simulated. Meanwhile, the model estimated that plant‐mediated transport, diffusion, and ebullition contributed to ∼23.5%, 15.0%, and 61.5% of CH 4 transport, respectively. A parameter sensitivity analysis showed that CH 4 substrate and CH 4 production were the most critical mechanisms regulating temporal patterns of surface CH 4 fluxes both under ambient conditions and warming treatments. This knowledge will be used to improve Earth system model predictions of these high‐carbon ecosystems from plot to regional scales.

58 GEOSCIENCES↗
DOE OSTI2020

Metagenomic and Metatranscriptomic Analyses Revealed Uncultured Bacteroidales Populations as the Dominant Proteolytic Amino Acid Degraders in Anaerobic Digesters

Current understanding of amino acid (AA) degraders in anaerobic digesters is mainly based on cultured species, whereas microorganisms that play important roles in a complex microbial community remain poorly characterized. This study investigated short-term enrichments degrading single AAs using metagenomics and metatranscriptomics. Metagenomic analysis revealed that populations related to cultured AA degraders had an abundance <2.5% of the sequences. In contrast, metagenomic-assembled bins related to uncultured Bacteroidales collectively accounted for >35% of the sequences. Phylogenetic analyses suggested that these Bacteroidales populations represented a yet-to-be characterized family lineage, i.e., Bacteroidetes vadinHA17. The bins possessed the genetic capacity related to protein degradation, including surface adhesion (3–7 genes), secreted peptidase (52–77 genes), and polypeptide-specific transporters (2–5 genes). Furthermore, metatranscriptomics revealed that these Bacteroidales populations expressed the complete metabolic pathways for degrading 16 to 17 types of AAs in enrichments fed with respective substrates. These characteristics were distinct from cultured AA degraders including Acidaminobacter and Peptoclostridium, suggesting the uncultured Bacteroidales were the major protein-hydrolyzing and AA-degrading populations. These uncultured Bacteroidales were further found to be dominant and active in full-scale anaerobic digesters, indicating their important ecological roles in the native habitats. “Candidatus Aminobacteroidaceae” was proposed to represent the previously uncharted family Bacteroidetes vadinHA17.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Spectroscopy-based isotopic (δ 13 C) analysis for high spatial resolution of carbon exchange in the rhizosphere

The rhizosphere is a highly dynamic zone bridging plant roots with needed nutrient resources in soil. While the rhizosphere may be small, it has a disproportionally large impact on plant success and biomass production. A suite of rhizosphere-hosted microbial and geochemical interactions facilitate nutrient acquisition by plant roots, and, in turn, the roots stimulate these processes by supplying organic carbon into the rhizosphere. The small physical dimensions of the rhizosphere, however, can constrain efforts to elucidate key carbon exchange processes and their spatial extent and localization. We present a method for spatially resolved δ 13 C analysis of rhizosphere samples by coupling laser ablation (LA) sampling with isotopic analysis using capillary absorption spectroscopy (CAS) which differs from conventional mass spectrometer (MS) approaches. The CAS system has high sensitivity (requires fewer nanomoles of CO 2 per analysis) than comparable MS systems, which enables reduced sample size requirements to thereby improve spatial resolution (from 25 μm to as low as a projected 5 μm spatial resolution). We demonstrate the utility of CAS using rhizosphere samples from switchgrass plants exposed to 13 CO 2 . As a result, this technique will provide a capability for tracking the extent and spatial distribution of root exudate into the rhizosphere at highly detailed spatial scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

Abstract Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides , is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides . The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2023

RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion

We report lignin-derived mixtures intended for bioconversion commonly contain high concentrations of aromatic acids, aliphatic acids, and salts. The inherent toxicity of these chemicals places a significant bottleneck upon the effective use of microbial systems for the valorization of these mixtures. Pseudomonas putida KT2440 can tolerate stressful quantities of several lignin-related compounds, making this bacterium a promising host for converting these chemicals to valuable bioproducts. Nonetheless, further increasing P. putida tolerance to chemicals in lignin-rich substrates has the potential to improve bioprocess performance. Accordingly, we employed random barcoded transposon insertion sequencing (RB-TnSeq) to reveal genetic determinants in P. putida KT2440 that influence stress outcomes during exposure to representative constituents found in lignin-rich process streams. The fitness information obtained from the RB-TnSeq experiments informed engineering of strains via deletion or constitutive expression of several genes. Namely, ΔgacAS, ΔfleQ, ΔlapAB, ΔttgR::P tac :ttgABC, Ptac:PP_1150:PP_1152, ..delta..relA, and ΔPP_1430 mutants showed growth improvement in the presence of single compounds, and some also exhibited greater tolerance when grown using a complex chemical mixture representative of a lignin-rich chemical stream. Overall, this work demonstrates the successful implementation of a genome-scale screening tool for the identification of genes influencing stress tolerance against notable compounds within lignin-enriched chemical streams, and the genetic targets identified herein offer promising engineering targets for improving feedstock tolerance in lignin valorization strains of P. putida KT2440.

09 BIOMASS FUELS↗
DOE OSTI2022

Spatial turnover of soil viral populations and genotypes overlain by cohesive responses to moisture in grasslands

Viruses shape microbial communities, food web dynamics, and carbon and nutrient cycling in diverse ecosystems. However, little is known about the patterns and drivers of viral community composition, particularly in soil, precluding a predictive understanding of viral impacts on terrestrial habitats. To investigate soil viral community assembly processes, here we analyzed 43 soil viromes from a rainfall manipulation experiment in a Mediterranean grassland in California. We identified 5,315 viral populations (viral operational taxonomic units [vOTUs] with a representative sequence ≥10 kbp) and found that viral community composition exhibited a highly significant distance–decay relationship within the 200-m 2 field site. This pattern was recapitulated by the intrapopulation microheterogeneity trends of prevalent vOTUs (detected in ≥90% of the viromes), which tended to exhibit negative correlations between spatial distance and the genomic similarity of their predominant allelic variants. Although significant spatial structuring was also observed in the bacterial and archaeal communities, the signal was dampened relative to the viromes, suggesting differences in local assembly drivers for viruses and prokaryotes and/or differences in the temporal scales captured by viromes and total DNA. Despite the overwhelming spatial signal, evidence for environmental filtering was revealed in a protein-sharing network analysis, wherein a group of related vOTUs predicted to infect actinobacteria was shown to be significantly enriched in low-moisture samples distributed throughout the field. Overall, our results indicate a highly diverse, dynamic, active, and spatially structured soil virosphere capable of rapid responses to changing environmental conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Impact of crowding on the diversity of expanding populations

Crowding effects critically impact the self-organization of densely packed cellular assemblies, such as biofilms, solid tumors, and developing tissues. When cells grow and divide, they push each other apart, remodeling the structure and extent of the population’s range. Recent work has shown that crowding has a strong impact on the strength of natural selection. However, the impact of crowding on neutral processes, which controls the fate of new variants as long as they are rare, remains unclear. Here, we quantify the genetic diversity of expanding microbial colonies and uncover signatures of crowding in the site frequency spectrum. By combining Luria–Delbrück fluctuation tests, lineage tracing in a novel microfluidic incubator, cell-based simulations, and theoretical modeling, we find that the majority of mutations arise behind the expanding frontier, giving rise to clones that are mechanically “pushed out” of the growing region by the proliferating cells in front. These excluded-volume interactions result in a clone-size distribution that solely depends on where the mutation first arose relative to the front and is characterized by a simple power law for low-frequency clones. Our model predicts that the distribution depends on a single parameter—the characteristic growth layer thickness—and hence allows estimation of the mutation rate in a variety of crowded cellular populations. Combined with previous studies on high-frequency mutations, our finding provides a unified picture of the genetic diversity in expanding populations over the whole frequency range and suggests a practical method to assess growth dynamics by sequencing populations across spatial scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Throughfall exclusion and fertilization effects on tropical dry forest tree plantations, a large-scale experiment

Abstract. Across tropical ecosystems, global environmental change is causing drier climatic conditions and increased nutrient deposition. Such changes represent large uncertainties due to unknown interactions between drought and nutrient availability in controlling ecosystem net primary productivity (NPP). Using a large-scale manipulative experiment, we studied for 4 years whether nutrient availability affects the individual and integrated responses of aboveground and belowground ecosystem processes to throughfall exclusion in 30-year-old mixed plantations of tropical dry forest tree species in Guanacaste, Costa Rica. We used a factorial design with four treatments: control, fertilization (F), drought (D), and drought + fertilization (D + F). While we found that a 13 %–15 % reduction in soil moisture only led to weak effects in the studied ecosystem processes, NPP increased as a function of F and D + F. The relative contribution of each biomass flux to NPP varied depending on the treatment, with woody biomass being more important for F and root biomass for D + F and D. Moreover, the F treatment showed modest increases in maximum canopy cover. Plant functional type (i.e., N fixation or deciduousness) and not the experimental manipulations was the main source of variation in tree growth. Belowground processes also responded to experimental treatments, as we found a decrease in nodulation for F plots and an increase in microbial carbon use efficiency for F and D plots. Our results emphasize that nutrient availability, more so than modest reductions in soil moisture, limits ecosystem processes in tropical dry forests and that soil fertility interactions with other aspects of drought intensity (e.g., vapor pressure deficit) are yet to be explored.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

CFD Simulation of Aerobic Gas Fermentation to Enable Commercial Conversion of CO 2 into Aquaculture and Animal Feed: Cooperative Research and Development (Final Report)

NovoNutrients’ fermentation technology uses energy from hydrogen to transform industrial CO2 emissions into premium animal feed ingredients and other valuable products. A single NovoNutrients’ commercial manufacturing plant will capture and convert over 200,000 tons/yr of CO2 into over 100,000 tons/yr of high-protein feed. Key to the rapid and widespread deployment of the technology is maximization of its productivity and energy efficiency. Robust, physically based computational models of the technology will significantly increase productivity and efficiency, accelerating NovoNutrients’ technology to manufacturing scale. NREL has unique capabilities for creating and running such computational models. NREL's existing aerobic bioreaction computational fluid dynamics (CFD) models will be adapted to NovoNutrients’ gas fermentation (CO2, H2, O2) technology. The multiphysics CFD simulations require thousands of high-performance computing (HPC) node hours to simulate the complex geometries and contents of NovoNutrients’ industrial bioreactors. The experimentally validated CFD models were used to identify optimally efficient and productive bioreactor designs and operating conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Novel Geochemistry Determined from High P-T Simulation Experiments of HFTS 2

A standard method in unconventional oil and gas production is the process of hydraulic fracturing followed by a shut-in period, during which the fracture fluid remains pressurized in the reservoir for up to three weeks before production begins. Despite this widely used process, very little is known about what occurs in the reservoir during this shut-in process. In order to properly delineate potential reservoir reactions that may occur during shut-in that would lead to corrosion and scaling events, experiments were conducted in high pressure, high temperature reactors to simulate conditions of the Wolfcamp Formation in the Delaware Basin. Experimental design allowed the assessment of the effect of proppant, microbiology, and time on the mineralogy and fluid chemistry in the reservoir. Results suggest the biggest impact on fluid chemistry and shale mineralogy during shut-in is time. Analyses demonstrate dissolution of the shale material, with maximum dissolved ions occurring after 7 days of the shut-in period. After 21 days, results suggest precipitation occurs. The Delaware Basin is demonstrated to be high in sulfate content, which further increases in the fluid due to dissolution reactions during shut-in. The pressure vessel experiments suggest there was no significant contribution to reactions from microbiology during the shut-in period. However, early production samples demonstrate a significant selection of the microorganism Caminicella, a genus of which has previously been correlated to corrosion and sulfide production. Results suggest shut-in conditions may provide high sulfate concentrations that could later be utilized by a shifted microbial community to drive potential well infrastructure failure. This is the first study to incorporate microbiology, mineralogy, and fluid chemistry to investigate the fundamental geochemical reactions that occur during shut-in and early phase production of the Delaware Basin. Results from this study can complement observations from the Hydraulic Fracture Test Site 2 observations.

Gulliver, Djuna↗
DOE OSTI2023

Microbial production of high octane and high sensitivity olefinic ester biofuels

Abstract Background Advanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety. Results Optimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose. Conclusion Our study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.

09 BIOMASS FUELS↗
DOE OSTI2023

Data for Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides . Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm–3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Conversion↗
DOE OSTI2024

Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bioaerosols as indicators of central Arctic ice nucleating particle sources

The Arctic is warming at a rapid rate, with implications for microbial communities as the ecosystems change. Some microbes and biogenic materials can affect the persistence of long-lived mixed-phase clouds by serving as ice nucleating particles (INPs). The presence of INPs modulates the cloud phase, and long-term measurements are important to elucidate their seasonal sources and to predict future change. The Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) expedition in 2019–2020 provided the first year-long measurements of bioaerosols and INPs in the central Arctic, with 3 d filters for amplicon sequencing and cumulative INP concentrations from −5 to −30 °C. Here, we investigated the INP seasonal cycle and its relation to the seasonal cycle of bacteria and eukaryotes. INPs were greatly elevated and compositionally similar in summer, aligning with a greater prevalence of local bioaerosol sources, but, despite this, a diverse mixture of sources (marine and terrestrial) was present all times. A common broader Arctic INP population is hypothesized for much of the year by comparable coincident data collected in Svalbard and a sensitivity of both the INPs and bioaerosols to large-scale events.

Barry, Kevin R. [Colorado State Univ., Fort Collin↗
DOE OSTI2020

Environmental controls on ecosystem-scale cold-season methane and carbon dioxide fluxes in an Arctic tundra ecosystem

Abstract. Understanding the processes that influence and control carbon cycling in Arctic tundra ecosystems is essential for making accurate predictions about what role these ecosystems will play in potential future climate change scenarios. Particularly, air–surface fluxes of methane and carbon dioxide are of interest as recent observations suggest that the vast stores of soil carbon found in the Arctic tundra are becoming more available to release to the atmosphere in the form of these greenhouse gases. Further, harsh wintertime conditions and complex logistics have limited the number of year-round and cold-season studies and hence too our understanding of carbon cycle processes during these periods. We present here a two-year micrometeorological data set of methane and carbon dioxide fluxes, along with supporting soil pore gas profiles, that provide near-continuous data throughout the active summer and cold winter seasons. Net emission of methane and carbon dioxide in one of the study years totalled 3.7 and 89 g C m−2 a−1 respectively, with cold-season methane emission representing 54 % of the annual total. In the other year, net emission totals of methane and carbon dioxide were 4.9 and 485 g C m−2 a−1 respectively, with cold-season methane emission here representing 82 % of the annual total – a larger proportion than has been previously reported in the Arctic tundra. Regression tree analysis suggests that, due to relatively warmer air temperatures and deeper snow depths, deeper soil horizons – where most microbial methanogenic activity takes place – remained warm enough to maintain efficient methane production whilst surface soil temperatures were simultaneously cold enough to limit microbial methanotrophic activity. These results provide valuable insight into how a changing Arctic climate may impact methane emission, and highlight a need to focus on soil temperatures throughout the entire active soil profile, rather than rely on air temperature as a proxy for modelling temperature–methane flux dynamics.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2006

Microbial Characterization of Internal Active Thermal Control System (IATCS) Hardware Surfaces after Five Years of Operation in the International Space Station

A flex hose assembly containing aqueous coolant from the International Space Station (ISS) Internal Active Thermal Control System (IATCS) consisting of a 2 foot section of Teflon hose and quick disconnects (QDs) and a Special Performance Checkout Unit (SPCU) heat exchanger containing separate channels of IATCS coolant and iodinated water used to cool spacesuits and Extravehicular Mobility Units (EMUS) were returned for destructive analyses on Shuttle return to flight mission STS-114. The original aqueous IATCS coolant used in Node 1, the Laboratory Module, and the Airlock consisted of water, borate (pH buffer), phosphate (corrosion control), and silver sulfate (microbiological control) at a pH of 9.5 +/- 0.5. Chemical changes occurred after on-orbit implementation including a decrease to pH 8.4 due to the diffusion of carbon dioxide through the Teflon hoses, an increase in nickel ions due to general corrosion of heat exchanger braze coatings, a decrease in phosphate concentration due to precipitation of nickel phosphate, and the rapid disappearance of silver ions due to deposition on hardware surfaces. Also associated with the coolant chemistry changes was an increase in planktonic microorganisms from less than 100 colony forming units (CFU) per 100 ml to approximately 1 million CFU per 100 ml. Attachment and growth of microorganisms to the system surfaces (biofilm) was suspected due to the levels of planktonic microorganisms in the coolant. Biofilms can reduce coolant flow, reduce heat transfer, amplify degradation of system materials initiated by chemical corrosion, and enhance mineral scale formation.

Roman, Monsi C.↗
DOE OSTI2023

Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm 3 ) capable of generating an electron density of greater than 10 13 cm –3 was constructed to produce reactive species (N 2 *, N 2 +, O, OH, and H α ) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. In conclusion, this compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

59 BASIC BIOLOGICAL SCIENCES↗