Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Microbial growth rate”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

EcoBOT: an AI/ML enabled automated phenotyping capability for model plants

Introduction: Advances in automation and AI/ML offer new opportunities for plant science, including design, modeling, and analysis. This study aimed to develop an automated platform for researching small model plants under axenic conditions and integrate it with AI/ML tools. Methods: The EcoBOT platform was developed, which consists of sterile containers (EcoFABs) for growing plants and imaging for monitoring plant growth and health. Brachypodium distachyon was grown on the EcoBOT, and its response to nutrient limitation and copper stress was evaluated. Results: The results showed that Brachypodium distachyon grown in the EcoBOT maintained sterility and responded to nutrient limitation and copper stress. Analysis of over 6,500 root and shoot images revealed varying sensitivity and response rates to copper. Bayesian Optimization was used to improve model accuracies relating copper concentrations to plant biomass via sequential experiments, resulting in a >30% improvement. Discussion: The findings of this study demonstrate the potential of the EcoBOT platform for researching plant responses to environmental factors. Future experiments could focus on relating other chemical stresses and microbial interactions to create generalized models of plant responses.

AI image analysis↗
NASA NTRS2004

Potential Application of Anaerobic Extremophiles for Hydrogen Production

During substrate fermentation many anaerobes produce the hydrogen as a waste product, which often regulates the growth of the cultures as an inhibitor. In nature the hydrogen is usually removed from the ecosystem due to its physical properties or by consumption of hydrogen by secondary anaerobes, which sometimes behave as competitors for electron donors as is seen in the classical example in anaerobic microbial communities via the interaction between methanogens and sulfate- or sulfur- reducers. It was demonstrated previously on mixed cultures of anaerobes at neutral pH that bacterial hydrogen production could provide an alternative energy source. But at neutral pH the original cultures can easily be contaminated by methanogens, a most unpleasant side effect of these conditions is the development of pathogenic bacteria. In both cases the rate of hydrogen production was dramatically decreased since some part of the hydrogen was transformed to methane, and the cultivation of human pathogens on a global scale is very dangerous. In our laboratory, experiments with obligately alkaliphilic bacteria that excrete hydrogen as the end metabolic product were performed at different temperature regimes. Mesophilic and moderately thermophilic bacterial cultures have been studied and compared for the most effective hydrogen production. For high-mineralized media with pH 9.5-10.0 not many methanogens are known to exist. Furthermore, the development of pathogenic contaminant microorganisms is virtually impossible: carbonate-saturated solutions are used as antiseptics in medicine. Therefore the cultivation of alkaliphilic hydrogen producing bacteria could be considered as most safe process for global Scale industry in future. Here we present experimental data on the rates of hydrogen productivity for mesophilic, alkaliphilic, obligately anaerobic bacterium Spirocheta americana ASpG1 and moderately thermophilic, alkaliphilic, facultative anaerobe Anoxybacillus pushchinoensis K1 and discuss the potential implications for alternative energy sources.

Pikuta, Elena V.↗
NASA NTRS2017

Preventing Precipitation in the ISS Urine Processor

The ISS Urine Processor Assembly (UPA) was initially designed to achieve 85% recovery of water from pretreated urine on ISS. Pretreated urine is comprised of crew urine treated with flush water, an oxidant (chromium trioxide), and an inorganic acid (sulfuric acid) to control microbial growth and inhibit precipitation. Unfortunately, initial operation of the UPA on ISS resulted in the precipitation of calcium sulfate at 85% recovery. This occurred because the calcium concentration in the crew urine was elevated in microgravity due to bone loss. The higher calcium concentration precipitated with sulfate from the pretreatment acid, resulting in a failure of the UPA due to the accumulation of solids in the Distillation Assembly. Since this failure, the UPA has been limited to a reduced recovery of water from urine to prevent calcium sulfate from reaching the solubility limit. NASA personnel have worked to identify a solution that would allow the UPA to return to a nominal recovery rate of 85%. This effort has culminated with the development of a pretreatment based on phosphoric acid instead of sulfuric acid. By eliminating the sulfate associated with the pretreatment, the brine can be concentrated to a much higher concentration before calcium sulfate reach the solubility limit. This paper summarizes the development of this pretreatment and the testing performed to verify its implementation on ISS.

Muirhead, Dean↗
DOE OSTI2022

Emiliania huxleyi —Bacteria Interactions under Increasing CO 2 Concentrations

The interactions established between marine microbes, namely phytoplankton–bacteria, are key to the balance of organic matter export to depth and recycling in the surface ocean. Still, their role in the response of phytoplankton to rising CO 2 concentrations is poorly understood. Here, we show that the response of the cosmopolitan Emiliania huxleyi (E. huxleyi) to increasing CO 2 is affected by the coexistence with bacteria. Specifically, decreased growth rate of E. huxleyi at enhanced CO 2 concentrations was amplified in the bloom phase (potentially also related to nutrient concentrations) and with the coexistence with Idiomarina abyssalis (I. abyssalis) and Brachybacterium sp. In addition, enhanced CO 2 concentrations also affected E. huxleyi’s cellular content estimates, increasing organic and decreasing inorganic carbon, in the presence of I. abyssalis, but not Brachybacterium sp. At the same time, the bacterial isolates only survived in coexistence with E. huxleyi, but exclusively I. abyssalis at present CO 2 concentrations. Bacterial species or group-specific responses to the projected CO 2 rise, together with the concomitant effect on E. huxleyi, might impact the balance between the microbial loop and the export of organic matter, with consequences for atmospheric carbon dioxide.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

A trait syndrome ties cell morphology to glycolysis across the yeast subphylum

Co-variation of traits provides fundamental insights into constraints governing their evolution. An inverse correlation between glucose uptake rates (GURs) and cell surface area-to-volume (SA:V) ratios across 11 yeast species was recently reported. Here, we expand the analysis to 282 species to assess the generalizability of this correlation across Saccharomycotina yeasts and the contribution of shared evolutionary history to the co-variation of these traits. Using phylogenetic regression models, we found extracellular acidification rates (ECARs, used as a proxy for GURs) were weakly, but significantly, correlated with SA:V across Saccharomycotina. ECARs were also correlated with genome sizes and growth rates. Our findings support the reported correlation between GURs and SA:V ratios, but suggest other associated traits, including genome sizes. Specifically, yeasts that consume glucose faster tend to have lower SA:V, faster growth rates, and larger genomes, suggesting a trait syndrome governing several metabolic, genomic, and morphological traits across the yeast subphylum.

biological sciences↗
DOE OSTI2022

Evaluating changes in growth and pigmentation of Cladosporium cladosporioides and Paecilomyces variotii in response to gamma and ultraviolet irradiation

Abstract Melanin-containing fungi (black molds) have the capacity to thrive under extreme environmental conditions such as the elevated radiation levels inside the former Chernobyl reactors. These fungi have been hypothesized to grow toward and use gamma radiation as an energy source, but the literature does not clearly address which energies of the electromagnetic spectrum, if any, positively affect fungal growth. The goal of this work was to characterize the response of non-melanized and melanized fungi to two distinct electromagnetic wavelengths, i.e., ultraviolet (UV) and gamma ray, keeping absorption and other potentially confounding variables constant. Exposure to UV or gamma radiation induced significant changes in fungi pigmentation, but not growth rate of Cladosporium cladosporioides and Paecilomyces variotii . Specifically, increased pigmentation of both fungi was observed in samples exposed to UV, while decreased pigmentation was observed for gamma-irradiated samples. These results provide new insights into the role of electromagnetic energies on growth of fungi and provide an impetus to examine additional energies and types of radiation to develop a fundamental understanding of this phenomenon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Gene Amplification, Laboratory Evolution, and Biosensor Screening Reveal MucK as a Terephthalic Acid Transporter in Acinetobacter baylyi ADP1

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

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 OSTI2021

Fungal and bacterial growth variation due to drought and nitrogen addition experimental treatments. Loma Ridge Experimental Project. 2010-2012

Terrestrial ecosystem models assume that microbial communities respond instantaneously, or are immediately resilient, to environmental change. Here we tested this assumption by quantifying the resilience of a leaf litter community to changes in precipitation or nitrogen availability. By manipulating composition within a global change experiment, we decoupled the legacies of abiotic parameters versus that of the microbial community itself. After one rainy season, more variation in fungal composition could be explained by the original microbial inoculum than the litterbag environment (18% versus 5.5% of total variation). This compositional legacy persisted for 3 years, when 6% of the variability in fungal composition was still explained by the microbial origin. In contrast, bacterial composition was generally more resilient than fungal composition. Microbial functioning (measured as decomposition rate) was not immediately resilient to the global change manipulations; decomposition depended on both the contemporary environment and rainfall the year prior. Finally, using metagenomic sequencing, we showed that changes in precipitation, but not nitrogen availability, altered the potential for bacterial carbohydrate degradation, suggesting why the functional consequences of the two experiments may have differed. Predictions of how terrestrial ecosystem processes respond to environmental change may thus be improved by considering the legacies of microbial communities. This data package includes ten csv files (five data files and their corresponding data dictionaries) and one file-level metadata excel file. Data files contains information about which plots were exposed to treatments related to drought and nitrogen, information about litter bags reciprocal transplants manipulation for water input and nitrogen, detail information about water addition, precipitation records, and litter variables collected. Data dictionary files include detail explanation for each column in the data files. The file-level metadata file describes each file mentioned above. All the analyses were done using the R software.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Adaptive responses of Trichlorobacter lovleyi to nitrite detoxification reveal overlooked contributions of Geobacterales to nitrate ammonification

Abstract Poorly understood microorganisms “short-circuit” the nitrogen cycle via the dissimilatory nitrate reduction to ammonium to retain the element in agricultural lands and stimulate crop productivity. The prevalence of Geobacterales closely related to Trichlorobacter lovleyi in nitrate ammonification hotspots motivated us to investigate adaptive responses contributing to ammonification rates in the laboratory type strain T. lovleyi SZ. Here, we describe the identification of tightly regulated pathways for efficient nitrate foraging and respiration with acetate, an important intermediate of organic matter degradation that Geobacterales efficiently assimilate and oxidize. Challenging the established dogma that high carbon/nitrate ratios stimulate the reduction of nitrate to ammonium, T. lovleyi doubled rapidly across a wide range of ratios provided nitrate concentrations were low enough to prevent the accumulation of the toxic nitrite intermediate. Yet, excess electrons during hydrogenotrophic growth alleviated nitrite toxicity and stimulated the reduction of nitrate to ammonium even under conditions of severe acetate limitation. These findings underscore the importance of nitrite toxicity in the ammonification of nitrate by Geobacterales and provide much needed mechanistic understanding of microbial adaptations contributing to soil nitrogen conservation. This information is critical to enhance the predictive value of genomic-based traits in environmental surveys and to guide strategies for sustainable management of nitrogen fertilization as well as mitigation of green-house emissions and agrochemical leaching from agricultural lands.

Environmental Sciences & Ecology↗
NASA NTRSDate not supplied

Microbial Mutualism in (Modeled) Microgravity: Measuring the Effects of A Spaceflight-Like Fluid Environment on Cross-Feeding Communities

Microorganisms grow differently in spaceflight than they do on Earth. While numerous factors likely contribute, one ubiquitous feature of the space environment is altered gravity - but because microbes are generally considered too small to detect gravity directly, they likely experience the effects of gravity via changes in their fluid environment, primarily a reduction in convective mixing. This theory is supported by evidence that individual microbial strains experience starvation and acid stress in microgravity. If it is true, then microgravity would also have measurable effects on microbes in mixed communities, because many interspecies interactions involve the exchange of soluble metabolites through their medium (cross-feeding). Specifically, cooperative cross-feeding communities would grow more slowly in microgravity, and this effect should be reproducible in ground-based rotational culture systems (rotating wall vessels, RWVs) designed to simulate the quiescent environment of microgravity. Here we present preliminary results of our efforts to test this hypothesis using using a model system of Eschericia coli and Salmonella enterica that grow only when they can exchange methionine and acetate through the medium. We report on the development of a novel laboratory system that allows us measure growth rates and species ratios in real-time using fluorescence spectrometry as the organisms grow in RWVs. We are able to track growth rates of individual species and the co-culture continuously with high temporal resolution for several hours, a substantial improvement over prior RWV systems that require destructive sampling. We vary the fidelity of the microgravity simulation by varying the rotation rate, and find that rotation rate affects growth rates of the community more than that of individual strains, consistent with the claim that RWVs can simulate a microgravity-like quiescent environment. While more work needs to be done to probe the mechanisms of this effect, we hope that our system will ultimately allow us to generate quantitative predictions about the effects of spaceflight on organisms that are tiny but essential to sustaining long-term human space exploration.

microgravity↗
DOE OSTI2024

germs-lab/PAPER_miscanthus_soil_response

Nutrient inputs influence the sustainability of bioenergy crop production through contemporary (shortly after addition) and legacy effects (persisting over years) on microbial nitrogen (N) and carbon cycling, which contribute to greenhouse gas emissions. However, the relative importance of contemporary and legacy effects and how that could vary by crop functional types is poorly understood. Considering its rhizomatous roots and perennial growth, we hypothesized that Miscanthus × giganteus (M×g) would be more sensitive to legacy N fertilization and the historical context of its environment than an annual crop like maize. To test this hypothesis, we examined the effects of legacy and contemporary N inputs on nitrous oxide (N2O) and carbon dioxide (CO2) emissions, as well as key N cycling genes in soils where M×g and maize were grown. A 150-day soil incubation experiment was conducted using soils from a long-term M×g and maize fertility experiment with three historic N fertilization rates (0, 112, and 336 kg N ha−1 year−1) and a contemporary amendment (60 mg N kg−1) with negative control (0 mg N kg−1). We observed significant increases in cumulative N2O emissions in Mxg soils relative to maize soils, particularly at higher legacy fertilization rates, while contemporary N had no significant effect. Bacterial amoA gene abundance, which plays a significant role in nitrification in nutrient-rich soils, also increased with higher legacy fertilization rates in M×g soils but was unaffected by the contemporary N. In maize soils, legacy and contemporary N did not significantly affect N2O emissions, but cumulative CO2 emissions and amoA gene abundance significantly increased. The abundances of norB genes were not significantly influenced by either legacy fertilization or contemporary N amendments in either soil. Our findings demonstrate the greater importance of fertilization history over contemporary N in mediating soil N2O emissions, particularly for perennial bioenergy crops.

Lee, Jaejin↗
NASA NTRS2003

Energy Requirements of Hydrogen-utilizing Microbes: A Boundary Condition for Subsurface Life

Microbial ecosystems based on the energy supplied by water-rock chemistry carry particular significance in the context of geo- and astrobiology. With no direct dependence on solar energy, lithotrophic microbes could conceivably penetrate a planetary crust to a depth limited only by temperature or pressure constraints (several kilometers or more). The deep lithospheric habitat is thereby potentially much greater in volume than its surface counterpart, and in addition offers a stable refuge against inhospitable surface conditions related to climatic or atmospheric evolution (e.g., Mars) or even high-energy impacts (e.g., early in Earth's history). The possibilities for a deep microbial biosphere are, however, greatly constrained by life s need to obtain energy at a certain minimum rate (the maintenance energy requirement) and of a certain minimum magnitude (the energy quantum requirement). The mere existence of these requirements implies that a significant fraction of the chemical free energy available in the subsurface environment cannot be exploited by life. Similar limits may also apply to the usefulness of light energy at very low intensities or long wavelengths. Quantification of these minimum energy requirements in terrestrial microbial ecosystems will help to establish a criterion of energetic habitability that can significantly constrain the prospects for life in Earth's subsurface, or on other bodies in the solar system. Our early work has focused on quantifying the biological energy quantum requirement for methanogenic archaea, as representatives of a plausible subsurface metabolism, in anoxic sediments (where energy availability is among the most limiting factors in microbial population growth). In both field and laboratory experiments utilizing these sediments, methanogens retain a remarkably consistent free energy intake, in the face of fluctuating environmental conditions that affect energy availability. The energy yields apparently required by methanogens in these sediment systems for sustained metabolism are about half that previously thought necessary. Lowered energy requirements would imply that a correspondingly greater proportion of the planetary subsurface could represent viable habitat for microorganisms.

Hoehler, Tori M.↗
DOE OSTI2024

Impacts of Legacy and Contemporary Nitrogen Inputs on N 2 O and CO 2 Emissions in Miscanthus and Maize Cultivated Soils

ABSTRACT Nutrient inputs influence the sustainability of bioenergy crop production through contemporary (shortly after addition) and legacy effects (persisting over years) on microbial nitrogen (N) and carbon cycling, which contribute to greenhouse gas emissions. However, the relative importance of contemporary and legacy effects and how that could vary by crop functional types is poorly understood. Considering its rhizomatous roots and perennial growth, we hypothesized that Miscanthus × giganteus (M×g) would be more sensitive to legacy N fertilization and the historical context of its environment than an annual crop like maize. To test this hypothesis, we examined the effects of legacy and contemporary N inputs on nitrous oxide (N 2 O) and carbon dioxide (CO 2 ) emissions, as well as key N cycling genes in soils where M×g and maize were grown. A 150‐day soil incubation experiment was conducted using soils from a long‐term M×g and maize fertility experiment with three historic N fertilization rates (0, 112, and 336 kg N ha −1 year −1 ) and a contemporary amendment (60 mg N kg −1 ) with negative control (0 mg N kg −1 ). We observed significant increases in cumulative N 2 O emissions in Mxg soils relative to maize soils, particularly at higher legacy fertilization rates, while contemporary N had no significant effect. Bacterial amo A gene abundance, which plays a significant role in nitrification in nutrient‐rich soils, also increased with higher legacy fertilization rates in M×g soils but was unaffected by the contemporary N. In maize soils, legacy and contemporary N did not significantly affect N 2 O emissions, but cumulative CO 2 emissions and amo A gene abundance significantly increased. The abundances of nor B genes were not significantly influenced by either legacy fertilization or contemporary N amendments in either soil. Our findings demonstrate the greater importance of fertilization history over contemporary N in mediating soil N 2 O emissions, particularly for perennial bioenergy crops.

09 BIOMASS FUELS↗
DOE OSTI2022

Lipo-Chitooligosaccharides Induce Specialized Fungal Metabolite Profiles That Modulate Bacterial Growth

Lipo-chitooligosaccharides (LCOs) are historically known for their role as microbial-derived signaling molecules that shape plant symbiosis with beneficial rhizobia or mycorrhizal fungi. Recent studies showing that LCOs are widespread across the fungal kingdom have raised questions about the ecological function of these compounds in organisms that do not form symbiotic relationships with plants. To elucidate the ecological function of these compounds, we investigate the metabolomic response of the ubiquitous human pathogen Aspergillus fumigatus to LCOs. Our metabolomics data revealed that exogenous application of various types of LCOs to A. fumigatus resulted in significant shifts in the fungal metabolic profile, with marked changes in the production of specialized metabolites known to mediate ecological interactions. Using network analyses, we identify specific types of LCOs with the most significant effect on the abundance of known metabolites. Extracts of several LCO-induced metabolic profiles significantly impact the growth rates of diverse bacterial species. These findings suggest that LCOs may play an important role in the competitive dynamics of non-plant-symbiotic fungi and bacteria. This study identifies specific metabolomic profiles induced by these ubiquitously produced chemicals and creates a foundation for future studies into the potential roles of LCOs as modulators of interkingdom competition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. Here, we applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L –1 nerolidol. Harnessing Oxford Nanopore’s long-read genomic sequencing, we reveal a potential genetic cause—the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L –1 nerolidol and ~0.96 g L –1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2017

Where Is the Upper Altitude Boundary of Earth's Biosphere? An Upcoming Stratosphere Exploration Mission

Earths atmosphere provides a relatively thin barrier to the severe conditions of space. Terrestrial microorganisms from our planets surface gets swept up into the atmosphere at rates which are influenced by meteorology, season, and variety of other regionally-dependent conditions. Residence time of microbes in the atmosphere, dispersal patterns, and metabolic activity in clouds are topics of special interest in tropospheric aerobiology. But what about even higher altitudes? A century of ocean exploration has allowed the scientific community to characterize marine life at every depth. Such knowledge was enabled by routine access, specifically, submersible vehicles with sample return systems. The outer reaches of Earth's atmosphere probably contain trace signatures of microbial life swept up from the surface, but high-altitude exploration (from 18-50 km above sea level) has been severely constrained by a shortage of reliable affordable sample acquisition systems. Thus, attempts to establish the upper altitude limits of Earths biosphere boundary remain unresolved. Previous conclusions based on observations of microbial growth and DNA signatures from samples collected in the upper atmosphere have resulted in extraordinary claims for the tenacity of life in the stratosphere. This altitude range can be regarded as a natural laboratory for examining the edge of habitability, a topic of central important to the field of astrobiology; in fact, stratosphere conditions (high UV light and low extremes of temperature, pressure, and relative humidity) closely resemble stressors associated with the surface of Mars. Moreover, low biomass detection instruments developed exploring Earths upper atmosphere can contribute to future strategies for life detection with spacecraft exploring other worlds. While pioneering, many past stratospheric microbiology studies have been qualitative in nature and lacking rigorous contamination control measurements to verify system sterility. We have designed, constructed, and recently flown a novel system for collecting upper atmosphere bioaerosols using the NASA C20A aircraft. We will report preliminary mission results from the lower stratosphere (characterizing abundance, diversity and survivability or airborne microorganisms), describe our methods for reducing false positives, and outline the open scientific engineering challenges in an ongoing effort to better establish Earths high-altitude biosphere boundary.

Smith, David J.↗