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DOE OSTI2021

Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection

The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biochemistry & Molecular Biology↗

NASA NTRS1992

Coupling plant growth and waste recycling systems in a controlled life support system (CELSS)

The development of bioregenerative systems as part of the Controlled Ecological Life Support System (CELSS) program depends, in large part, on the ability to recycle inorganic nutrients, contained in waste material, into plant growth systems. One significant waste (resource) stream is inedible plant material. This research compared wheat growth in hydroponic solutions based on inorganic salts (modified Hoagland's) with solutions based on the soluble fraction of inedible wheat biomass (leachate). Recycled nutrients in leachate solutions provided the majority of mineral nutrients for plant growth, although additions of inorganic nutrients to leachate solutions were necessary. Results indicate that plant growth and waste recyling systems can be effectively coupled within CELSS based on equivalent wheat yield in leachate and Hoagland solutions, and the rapid mineralization of waste organic material in the hydroponic systems. Selective enrichment for microbial communities able to mineralize organic material within the leachate was necessary to prevent accumulation of dissolved organic matter in leachate-based solutions. Extensive analysis of microbial abundance, growth, and activity in the hydroponic systems indicated that addition of soluble organic material from plants does not cause excessive microbial growth or 'biofouling', and helped define the microbially-mediated flux of carbon in hydroponic solutions.

Garland, Jay L.↗

NASA NTRS2000

Measures of Water Quality in Merrit Island National Wildlife Refuge Impoundments and Adjacent Indian River Lagoon

The goal of this project was to conduct preliminary investigations to determine appropriate sampling strategies to measure the flux of dissolved nutrients (specifically, NH4+, NO3-, NO2-, and PO4(3-)) and suspended particulate matter (TSS) between impoundments and the IRL in preparation for an intensive three-year monitoring program. In addition to nutrients and TSS, a variety of common water quality indicators were also measured during these preliminary studies. Six impoundments and a single restored marsh were selected for study. Over a month long period, water samples were collected weekly at selected impoundment culverts. Water was collected in duplicate as independent grab samples from both the lagoon side and within the perimeter ditch directly adjacent to the culverts. Water quality indicators inside and outside the marsh impoundments were different. Ammonium, salinity, bacteria, and chlorophyll-a were higher inside the impoundments as expected possibly as a result of the great affect of evaporation on impoundment water. Water quality indicators responded rapidly both inside and outside the impoundments as exemplified by the increase in NH4(+)-N concentrations during a horseshoe crab die-off. Water quality indicators were high variable during the month in which water samples were collected. Because the impoundments are widely spaced it is logistically unrealistic to sample each of the impoundments and associated seagrass beds on a single day, sampling must be stratified to allow patterns of material movement and the annual flux of materials to and from the impoundments to be determined.

Blum, Linda K.↗

DOE OSTI2021

Effects of salinity and nutrients on metabolism and growth of Ulva lactuca : Implications for bioremediation of coastal watersheds

We studied Ulva lactuca to determine its potential for bioremediation of coastal watersheds. We cultured Ulva in orthogonal combinations of two salinities and three nutrient concentrations for six weeks, and then measured its growth, photosynthesis, chlorophyll fluorescence, nitrogen, carbon and phosphorus tissue concentrations, and carbon and nitrogen uptake pathways. Our findings show that Ulva was negatively affected by decreased salinity but these effects were ameliorated by the addition of nutrients to the water, such as would be expected from freshwater runoff during heavy rain events. Also, increased nutrients resulted in altered nitrogen (NH$_4^ +$ vs. NO$_3^-$) and carbon (HCO$_3^-$ vs. CO 2 ) uptake pathways, which can allow Ulva to retain its bloom potential even under reduced salinities. Together, our study suggests that Ulva is an ideal species to grow for the purpose of bioremediation of coastal bays and estuaries, even during storms that freshen the surface waters and increase nutrients.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates

Abstract Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24–48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

3D printing↗

DOE OSTI2025

Oceanic mixing and waves in the presence of a suspended canopy

Large-eddy simulations are analysed to determine the influence of suspended canopies, such as those formed in macroalgal farms, on ocean mixed layer (OML) deepening and internal wave generation. In the absence of a canopy, we show that Langmuir turbulence, when compared with wind-driven shear turbulence, results in a deeper OML and more pronounced internal waves beneath the OML. Subsequently, we examine simulations with suspended canopies of varying densities located in the OML, in the presence of a background geostrophic current. Intensified turbulence occurs in the shear layer at the canopy’s bottom edge, arising from the interaction between the geostrophic current and canopy drag. Structures resembling Kelvin–Helmholtz (KH) instability emerge as the canopy shear layer interacts with the underlying stratification, radiating internal waves beneath the OML. Both intensified turbulence and lower-frequency motions associated with KH-type structures are critical factors in enhancing mixing. Consequently, the OML depth increases by up to a factor of two compared with cases without a canopy. Denser canopies and stronger geostrophic currents lead to more pronounced KH-type structures and internal waves, stronger turbulence and greater OML deepening. Additionally, vertical nutrient transport is enhanced as the OML deepens due to the presence of the canopy. Considering that the canopy density investigated in this study closely represents offshore macroalgal farms, these findings suggest a mechanism for passive nutrient entrainment conducive to sustainable farming. Overall, this study reveals the intricate interactions between the suspended canopy, turbulent mixing and stratification, underscoring their importance in reshaping OML characteristics.

Bo, Tong (ORCID:0000000260300561)↗

DOE OSTI2023

Versatile microbial communities rapidly assimilate ammonium hydroxide-treated plastic waste

Abstract Waste plastic presently accumulates in landfills or the environment. While natural microbial metabolisms can degrade plastic polymers, biodegradation of plastic is very slow. This study demonstrates that chemical deconstruction of polyethylene terephthalate (PET) with ammonium hydroxide can replace the rate limiting step (depolymerization) and by producing plastic-derived terephthalic acid and terephthalic acid monoamide. The deconstructed PET (DCPET) is neutralized with phosphoric acid prior to bioprocessing, resulting in a product containing biologically accessible nitrogen and phosphorus from the process reactants. Three microbial consortia obtained from compost and sediment degraded DCPET in ultrapure water and scavenged river water without addition of nutrients. No statistically significant difference was observed in growth rate compared to communities grown on DCPET in minimal culture medium. The consortia were dominated by Rhodococcus spp., Hydrogenophaga spp., and many lower abundance genera. All taxa were related to species known to degrade aromatic compounds. Microbial consortia are known to confer flexibility in processing diverse substrates. To highlight this, we also demonstrate that two microbial consortia can grow on similarly deconstructed polyesters, polyamides, and polyurethanes in water instead of medium. Our findings suggest that microbial communities may enable flexible bioprocessing of mixed plastic wastes when coupled with chemical deconstruction.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Long-term nitrogen loading alleviates phosphorus limitation in terrestrial ecosystems

Increased human-derived nitrogen (N) deposition to terrestrial ecosystems has resulted in widespread phosphorus (P) limitation of net primary productivity. However, it remains unclear if and how N-induced P limitation varies over time. Soil extracellular phosphatases catalyze the hydrolysis of P from soil organic matter, an important adaptive mechanism for ecosystems to cope with N-induced P limitation. In this study we show, using a meta-analysis of 140 studies and 668 observations worldwide, that N stimulation of soil phosphatase activity diminishes over time. Whereas short-term N loading (≤5 years) significantly increased soil phosphatase activity by 28%, long-term N loading had no significant effect. Nitrogen loading did not affect soil available P and total P content in either short- or long-term studies. Together, these results suggest that N-induced P limitation in ecosystems is alleviated in the long-term through the initial stimulation of soil phosphatase activity, thereby securing P supply to support plant growth. Our results suggest that increases in terrestrial carbon uptake due to ongoing anthropogenic N loading may be greater than previously thought.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennes(see United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metagenomic Insights Into the Microbial Iron Cycle of Subseafloor Habitats

Microbial iron cycling influences the flux of major nutrients in the environment (e.g., through the adsorptive capacity of iron oxides) and includes biotically induced iron oxidation and reduction processes. The ecological extent of microbial iron cycling is not well understood, even with increased sequencing efforts, in part due to limitations in gene annotation pipelines and limitations in experimental studies linking phenotype to genotype. This is particularly true for the marine subseafloor, which remains undersampled, but represents the largest contiguous habitat on Earth. To address this limitation, we used FeGenie, a database and bioinformatics tool that identifies microbial iron cycling genes and enables the development of testable hypotheses on the biogeochemical cycling of iron. Herein, we survey the microbial iron cycle in diverse subseafloor habitats, including sediment-buried crustal aquifers, as well as surficial and deep sediments. We inferred the genetic potential for iron redox cycling in 32 of the 46 metagenomes included in our analysis, demonstrating the prevalence of these activities across underexplored subseafloor ecosystems. We show that while some processes (e.g., iron uptake and storage, siderophore transport potential, and iron gene regulation) are near-universal, others (e.g., iron reduction/oxidation, siderophore synthesis, and magnetosome formation) are dependent on local redox and nutrient status. Additionally, we detected niche-specific differences in strategies used for dissimilatory iron reduction, suggesting that geochemical constraints likely play an important role in dictating the dominant mechanisms for iron cycling. Overall, our survey advances the known distribution, magnitude, and potential ecological impact of microbe-mediated iron cycling and utilization in sub-benthic ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. As a result, these protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1974

Arthrobacter globiformis and its bacteriophage in soil

An attempt was made to correlate bacteriophages for Arthrobacter globiformis with soils containing that bacterium. The phages were not detected unless the soil was nutritionally amended (with glucose or sucrose) and incubated for several days. Phage was continuously produced after amendment without the addition of host Arthrobacter. These results indicate that the bacteriophage is present in a masked state and that the bacteria are present in an insensitive form which becomes sensitive after addition of nutrient.

Casida, L. E., Jr.↗

NASA NTRS1981

A search for a nonbiological explanation of the Viking Labeled Release life detection experiment

The possibility of nonbiological reactions involving hydrogen peroxide being the source of the positive response detected by the Viking Labeled Release (LR) life detection experiment on the surface of Mars is assessed. Labeled release experiments were conducted in the LR Test Standards Module which replicates the Viking flight instrument configuration on analog Martian soils prepared to match the Viking inorganic analysis of Mars surface material to which an aqueous solution of hydrogen peroxide had been added. Getter experiments were also conducted to compare several reactions simultaneously in the presence and absence of UV radiation prior to the addition of nutrient. Hydrogen peroxide on certain analog soils is found to be capable of reproducing the kinetics and thermal information contained in the Mars data. The peroxide concentration necessary for this response, however, is shown to require a chemical stability or production rate much greater than seems likely in the Mars environment. As previous experiments have shown hydrogen peroxide to be the most likely nonbiological source of the positive LR response, it is concluded that the presence of a biological agent on Mars must not yet be ruled out.

Levin, G. V.↗

NASA NTRS2010

Needs Assessment for the Use of NASA Remote Sensing Data in the Development and Implementation of Estuarine and Coastal Water Quality Standards

The goals of the project are to provide information from satellite remote sensing to support numeric nutrient criteria development and to determine data processing methods and data quality requirements to support nutrient criteria development and implementation. The approach is to identify water quality indicators that are used by decision makers to assess water quality and that are related to optical properties of the water; to develop remotely sensed data products based on algorithms relating remote sensing imagery to field-based observations of indicator values; to develop methods to assess estuarine water quality, including trends, spatial and temporal variability, and seasonality; and to develop tools to assist in the development and implementation of estuarine and coastal nutrient criteria. Additional slides present process, criteria development, typical data sources and analyses for criteria process, the power of remote sensing data for the process, examples from Pensacola Bay, spatial and temporal variability, pixel matchups, remote sensing validation, remote sensing in coastal waters, requirements for remotely sensed data products, and needs assessment. An additional presentation examines group engagement and information collection. Topics include needs assessment purpose and objectives, understanding water quality decision making, determining information requirements, and next steps.

Spiering, Bruce↗

NASA NTRS2014

NASA Bioculture System: From Experiment Definition to Flight Payload

Starting in 2015, the NASA Bioculture System will be available to the science community to conduct cell biology and microbiology experiments on ISS. The Bioculture System carries ten environmentally independent Cassettes, which house the experiments. The closed loop fluids flow path subsystem in each Cassette provides a perfusion-based method for maintain specimen cultures in a shear-free environment by using a biochamber based on porous hollow fiber bioreactor technology. Each Cassette contains an incubator and separate insulated refrigerator compartment for storage of media, samples, nutrients and additives. The hardware is capable of fully automated or manual specimen culturing and processing, including in-flight experiment initiation, sampling and fixation, up to BSL-2 specimen culturing, and the ability to up to 10 independent cultures in parallel for statistical analysis. The incubation and culturing of specimens in the Bioculture System is a departure from standard laboratory culturing methods. Therefore, it is critical that the PI has an understanding the pre-flight test required for successfully using the Bioculture System to conduct an on-orbit experiment. Overall, the PI will conduct a series of ground tests to define flight experiment and on-orbit implementation requirements, verify biocompatibility, and determine base bioreactor conditions. The ground test processes for the utilization of the Bioculture System, from experiment selection to flight, will be reviewed. Also, pre-flight test schedules and use of COTS ground test equipment (CellMax and FiberCell systems) and the Bioculture System will be discussed.

Bioculture System↗

DOE OSTI2025

Controlled electrochemical nutrient delivery to enhance marine primary productivity

Oceanic photosynthesis contributes to approximately half of the Earth’s net annual primary productivity. Marine photosynthetic productivity has a high degree of heterogeneity due to spatial and temporal co-limitations of light, temperature, and/or nutrients. Across coastal, near-shore, and open ocean regions, insufficient concentrations of key nutrients (e.g., N, P, Fe) can limit primary productivity. Although studies have shown a significant increase in primary productivity with the addition of low doses of trace nutrients, a sustainable approach to reliably deliver and maintain low doses of nutrients and ensure their bioavailability, remains a challenge. Chemical nutrient addition has relied on the use of chelating agents to ensure nutrient bioavailability, but synthetic chelators are persistent environmental pollutants. In this study, we demonstrate for the first time the use of a controlled electrochemical nutrient delivery (CEND) approach to accelerate the growth of phytoplankton without the need for such chelators. Our study uses commercial stainless-steel electrodes to deliver low concentrations of iron to enhance growth rates in the microalga Picochlorum celeri TG2. To demonstrate the process control offered by the CEND method, we evaluate iron delivery as a function of pulse time, pulse frequency, and rest time between pulses. Our data show that at the same total Fe dose of 163 ppb, electrochemical iron delivery can achieve 9.54 ± 0.58 mg biomass/µg Fe, which is comparable to 9.14 ± 0.17 mg biomass/µg Fe achieved with chemical iron additions that include the synthetic chelating agent ethylenediaminetetraacetic acid (EDTA). Further, when different total iron doses (163 ppb, 325 ppb, and 650 ppb) were delivered over 72 h using CEND, biomass yield per iron dose was higher at lower doses: 9.54 ± 0.58 mg biomass/µg Fe at 163 ppb vs. 4.32 ± 0.32 mg biomass/µg Fe at 650 ppb. This highlights the benefits of CEND in delivering frequent and low doses of nutrients for improved process efficiency. Preliminary assessments show both lower cost and reduced greenhouse gas emissions from electrochemical over chemical iron additions with EDTA. The CEND approach opens new pathways to enhance marine primary productivity, without the unintended environmental impacts of synthetic chelators.

analytical chemistry↗

NASA NTRS1977

Recent results from the Viking Labeled Release experiment on Mars

The Labeled Release (LR) life detection experiment seeks detection of heterotrophic metabolism by monitoring radio-active gas evolution following the addition of a radioactive nutrient containing seven C-14 labeled organic substrates to surface material. LR results obtained on Mars prior to conjunction showed rapid evolution of radioactive counts upon addition of the nutrient to a fresh surface sample. The responses at both landing sites were quite similar. The additional results summarized in the present paper provide further information on the gas kinetics following a second injection of nutrient and on the effect of 'cold sterilization' of the Martian surface material. Specifically, it is shown that the production of gas from the LR nutrient is remarkably uniform; after reaction approaches completion, addition of more nutrient results in a net loss of the radioactive gas; the reactant in the Mars soil is completely inhibited by heating the soil to 160 C, and is largely inhibited by heating to 46 C. In contrast, exposure to 18 C for 2 sol (1 sol = 24.6 hr) does not appear to inhibit the reaction.

Levin, G. V.↗

NASA NTRSDate not supplied

Update to Developing a Multi-bioreactor Test Stand for the Evaluation of Biofilm Mitigation Technologies

Biofilm formation in spacecraft wastewater systems is a complex and impactful problem. Biomass flowing in these wastewater systems can cause clogging, higher differential pressures, and flow restrictions impacting equipment reliability and function. To explore this issue, the Marshall Space Flight Center’s biofilm test stand was created and has been utilized to examine multiple biofilm mitigation techniques over a long duration test time. This paper is an update to a previous paper, ICES-2021-122, and will serve to discuss the modifications and enhancements to the biofilm test stand. While the previous configuration tested heat treatments and coatings, this phase of testing will examine differing materials, competing bacteria provided by an external bioreactor, a prototype phytoremediation unit, and additional testing with a nutrient filter. Configuration changes include the addition of float switches, adjustment of the drain assembly architecture and additional support hardware for the competing bacteria bioreactor and phytoremediation units.

ECLSS↗