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DOE OSTI2023

Microbial sensitivity to temperature and sulfate deposition modulates greenhouse gas emissions from peat soils

Abstract Peatlands are among the largest natural sources of atmospheric methane (CH 4 ) worldwide. Microbial processes play a key role in regulating CH 4 emissions from peatland ecosystems, yet the complex interplay between soil substrates and microbial communities in controlling CH 4 emissions as a function of global change remains unclear. Herein, we performed an integrated analysis of multi‐omics data sets to provide a comprehensive understanding of the molecular processes driving changes in greenhouse gas (GHG) emissions in peatland ecosystems with increasing temperature and sulfate deposition in a laboratory incubation study. We sought to first investigate how increasing temperatures (4, 21, and 35°C) impact soil microbiome–metabolome interactions; then explore the competition between methanogens and sulfate‐reducing bacteria (SRBs) with increasing sulfate concentrations at the optimum temperature for methanogenesis. Our results revealed that peat soil organic matter degradation, mediated by biotic and potentially abiotic processes, is the main driver of the increase in CO 2 production with temperature. In contrast, the decrease in CH 4 production at 35°C was linked to the absence of syntrophic communities and the potential inhibitory effect of phenols on methanogens. Elevated temperatures further induced the microbial communities to develop high growth yield and stress tolerator trait‐based strategies leading to a shift in their composition and function. On the other hand, SRBs were able to outcompete methanogens in the presence of non‐limiting sulfate concentrations at 21°C, thereby reducing CH 4 emissions. At higher sulfate concentrations, however, the prevalence of communities capable of producing sufficient low‐molecular‐weight carbon substrates for the coexistence of SRBs and methanogens was translated into elevated CH 4 emissions. The use of omics in this study enhanced our understanding of the structure and interactions among microbes with the abiotic components of the system that can be useful for mitigating GHG emissions from peatland ecosystems in the face of global change.

AminiTabrizi, Roya↗

DOE OSTI2020

DISCOVR: Development of Integrated Screening, Cultivar Optimization, and Verification Research

The DISCOVR consortium has developed and is applying an integrated platform and workflow for standardized, deep characterization of highly productive and resilient microalgae strains. New strains are screened using three consecutive tiers in a conceptual down-selection funnel to arrive at the most promising strains. At tier I, strains are screened on gradient incubators to determine their temperature and salinity tolerance range, and to evaluate their resistance to infectious agents (e.g., chytrids). At tier II, the strains’ winter and summer season biomass productivities are quantified in Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, and biomass composition is determined under N-replete and N-deplete conditions. At tier III, the best winter and summer season strains are evaluated in indoor crash ponds to determine their resilience to induced infections, and strains are tested in outdoor raceway ponds to confirm high biomass productivity and culture stability. The top strains are then evaluated in seasonal SOT outdoor pond culture trials at AzCATI, the dedicated Department of Energy SOT (State of Technology) testbed. Additional research is carried out on evaluating growth promoting molecules, reducing the impact of oxygen inhibition, optimizing pond operational conditions, measuring compositional shift dynamics, developing integrated pest management strategies, analysing culture health using 96 well-plate tools, implementing spectroradiometric monitoring and using machine learning to predict culture crashes.

Huesemann, Michael H↗

DOE OSTI2023

Substrate-independent expression of key functional genes in Cycloclasticus pugetii strain PS-1 limits their use as markers for PAH biodegradation

Microbial degradation of petroleum hydrocarbons is a crucial process for the clean-up of oil-contaminated environments. Cycloclasticus spp. are well-known polycyclic aromatic hydrocarbon (PAH) degraders that possess PAH-degradation marker genes including rhd3α, rhd2α, and pahE. However, it remains unknown if the expression of these genes can serve as an indicator for active PAH degradation. Here, we determined transcript-to-gene (TtG) ratios with (reverse transcription) qPCR in cultures of Cycloclasticus pugetii strain PS-1 grown with naphthalene, phenanthrene, a mixture of these PAHs, or alternate substrates (i.e., no PAHs). Mean TtG ratios of 1.99 × 10 −2 , 1.80 × 10 −3 , and 3.20 × 10 −3 for rhd3α, rhd2α, and pahE, respectively, were measured in the presence or absence of PAHs. The TtG values suggested that marker-gene expression is independent of PAH degradation. Measurement of TtG ratios in Arctic seawater microcosms amended with water-accommodated crude oil fractions, and incubated under in situ temperature conditions (i.e., 1.5°C), only detected Cycloclasticus spp. rhd2α genes and transcripts (mean TtG ratio of 4.15 × 10 −1 ). The other marker genes—rhd3α and pahE—were not detected, suggesting that not all Cycloclasticus spp. carry these genes and a broader yet-to-be-identified repertoire of PAH-degradation genes exists. The results indicate that the expression of PAH marker genes may not correlate with PAH-degradation activity, and transcription data should be interpreted cautiously.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1974

Bio-conversion of water hyacinths into methane gas, part 1

Bio-gas and methane production from the microbial anaerobic decomposition of water hyacinths (Eichhornia crassipes) (Mart) Solms was investigated. These experiments demonstrated the ability of water hyacinths to produce an average of 13.9 ml of methane gas per gram of wet plant weight. This study revealed that sample preparation had no significant effect on bio-gas and/or methane production. Pollution of water hyacinths by two toxic heavy materials, nickel and cadmium, increased the rate of methane production from 51.8 ml/day for non-contaminated plants incubated at 36 C to 81.0 ml/day for Ni-Cd contaminated plants incubated at the same temperature. The methane content of bio-gas evolved from the anaerobic decomposition of Ni-Cd contaminated plants was 91.1 percent as compared to 69.2 percent methane content of bio-gas collected from the fermentation of non-contaminated plants.

Wolverton, B. C.↗

NASA NTRSDate not supplied

Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis

The cyanobacteria Arthrospira platensis NIES-39, commonly known as spirulina, could provide a fresh supply of nutrients for crew on long-duration spaceflight missions. Spirulina is a readily digestible food that is high in protein with all essential amino acids as well as significant levels of B vitamins, antioxidants, and anti-inflammatory metabolites. Spaceflight has multiple abiotic stressors such as increased ionizing radiation and microgravity, which causes a lack of convective mixing. These environmental conditions may impact productivity, nutritional composition, and in long-duration propagation, spaceflight stress may impact the genetic stability of spirulina cultures. We are developing an International Space Station experiment to continuously culture A. platensis for six months. Multi-omics profiling will be used to monitor for changes in the genome, transcriptome, proteome, and metabolome to determine if A. platensis is a suitable nutritional supplement on long-duration missions. During preflight testing we developed a protocol for inoculated liquid cultures to survive a 10-week storage period prior to photo-incubation. The bioreactor bag, temperature, and lighting conditions that support a 14-day growth cycle between passages were also determined. Media testing identified minimal salts supporting robust growth that can be stored in liquid or dry form. A simple filtration method was developed to dewater cultures and harvest biomass for frozen sample return. We optimized a cryopreservation method to enable return of live cells for isolation of individual A. platensis clones. The concept of operations for Space Algae-2 developed from these test results as well as progress on multi-omics analysis methods will be presented.

Algae↗

NASA NTRSDate not supplied

Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis

Algae↗

DOE OSTI2022

Substrate availability and not thermal acclimation controls microbial temperature sensitivity response to long-term warming

Microbes are responsible for cycling carbon (C) through soils, and predicted changes in soil C stocks under climate change are highly sensitive to shifts in the mechanisms assumed to control the microbial physiological response to warming. Two mechanisms have been suggested to explain the long-term warming impact on microbial physiology: microbial thermal acclimation and changes in the quantity and quality of substrates available for microbial metabolism. Yet studies disentangling these two mechanisms are lacking. To resolve the drivers of changes in microbial physiology in response to long-term warming, we sampled soils from 13- and 28-year-old soil warming experiments in different seasons. We performed short-term laboratory incubations across a range of temperatures to measure the relationships between temperature sensitivity of physiology (growth, respiration, carbon use efficiency, and extracellular enzyme activity) and the chemical composition of soil organic matter. We observed apparent thermal acclimation of microbial respiration, but only in summer, when warming had exacerbated the seasonally-induced, already small dissolved organic matter pools. Irrespective of warming, greater quantity and quality of soil carbon increased the extracellular enzymatic pool and its temperature sensitivity. We propose that fresh litter input into the system seasonally cancels apparent thermal acclimation of C-cycling processes to decadal warming. Our findings reveal that long-term warming has indirectly affected microbial physiology via reduced C availability in this system, implying that earth system models including these negative feedbacks may be best suited to describe long-term warming effects on these soils.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Functional capacities of microbial communities to carry out large scale geochemical processes are maintained during ex situ anaerobic incubation

Mechanisms controlling CO 2 and CH 4 production in wetlands are central to understanding carbon cycling and greenhouse gas exchange. However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO 2 and CH 4 dynamics.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1971

Microbiologic assay of space hardware.

Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

Favero, M. S.↗

NASA NTRS1974

Hazard analysis of Clostridium perfringens in the Skylab Food System

The Skylab Food System presented unique microbiological problems because food was warmed in null-gravity and because the heat source was limited to 69.4 C (to prevent boiling in null-gravity). For these reasons, the foods were manufactured using critical control point techniques of quality control coupled with appropriate hazard analyses. One of these hazard analyses evaluated the threat from Clostridium perfringens. Samples of food were inoculated with C. perfringens and incubated for 2 h at temperatures ranging from 25 to 55 C. Generation times were determined for the foods at various temperatures. Results of these tests were evaluated taking into consideration: food-borne disease epidemiology, the Skylab food manufacturing procedures, and the performance requirements of the Skylab Food System. Based on this hazard analysis, a limit for C. perfringens of 100/g was established for Skylab foods.

Bourland, C. T.↗

NASA NTRS1996

Fecundity of Quail in Spacelab Microgravity

Flight experiments in which fertilized Japanese quail eggs were allowed to develop to various ages in space, and the results of the following laboratory tests are described. Laboratory-based experiments concerned with the embryonic development of Japanese quail in gravity using simulated vibrations and G-force are reported. Effect of turning and ambient temperature at various days of incubation on the development of Japanese quail, and method to feed and water adult and newly hatched Japanese quail in microgravity using a gelatin-based diet as a solid water supply, are also described.

Wentworth, B. C.↗

NASA NTRS2021

BioSensor Users' Guide

The BioSensor is a fully autonomous 3-color LED-based spectrophotometer paired with a fluidics system that supports microbes in liquid culture. Originally developed for the BioSentinel CubeSat mission to study the response of a wild type and mutant strain of yeast to the deep space environment, the BioSensor consists of a series of 16-well, independently plumbed fluidics cards. The cards utilize filters to allow the fluidic system to administer reagents while constraining the microbes in their wells. Heaters on each card incubate to the appropriate growth temperature once the experiment begins. During the active experiment, the LED/detector system measures the transmission of light through each well at three specific wavelengths, similar to a standard benchtop spectrophotometer. The transmission/absorbance kinetics curves for each well are telemetered back to Earth, along with temperature data, for analysis on the ground. The BioSensor is being upgraded from its original CubeSat free-flyer interface to be a secondary payload on lunar landers, Gateway, and other BLEO applications, while maintaining the same functionality and science utility for future experiments.

Matthew Lera↗

NASA NTRS2021

BioSensor Users' Guide

The BioSensor is a fully autonomous 3-color LED-based spectrophotometer paired with a fluidics system that supports microbes in liquid culture. Originally developed for the BioSentinel CubeSat mission to study the response of a wild type and mutant strain of yeast to the deep space environment, the BioSensor consists of a series of 16-well, independently plumbed fluidics cards. The cards utilize filters to allow the fluidic system to administer reagents while constraining the microbes in their wells. Heaters on each card incubate to the appropriate growth temperature once the experiment begins. During the active experiment, the LED/detector system measures the transmission of light through each well at three specific wavelengths, similar to a standard benchtop spectrophotometer. The transmission/ absorbance kinetics curves for each well are telemetered back to Earth, along with temperature data, for analysis on the ground. The BioSensor is being upgraded from its original CubeSat free-flyer interface to be a secondary payload on lunar landers, Gateway, and other BLEO applications, while maintaining the same functionality and science utility for future experiments.

Matthew Lera↗

NASA NTRS1993

A circadian rhythm of conidiation in Neurospora crassa (L-12)

Two fungi growth chambers containing six growth tubes each are used in this experiment. One chamber is for the space experiment; the other is for the simultaneous ground control experiment. The hyphae of Neurospora crassa band A mutant are inoculated at one end of each tube. Both the chambers are kept at 3 C plus or minus 1.5 C to stop hyphae growth until the Spacelab is activated. After the activation, each chamber is transferred simultaneously to the Spacelab and a phytotron in KSC and kept in continuous light at the same temperature. After about 24 hours of light exposure, each chamber is inserted into a growth chamber bag to keep it in constant darkness. The circadian rhythm of conidiation is initiated by this light to dark transition. After the dark incubation for 5 days at room temperature, both the growth chambers are kept at 3 C plus or minus 1.5 C to stop growth of the hyphae. After the space shuttle lands, both conidiation patterns are compared and analyzed. It has been known that numerous physiological phenomena show circadian rhythms. They are characterized by the fact that the oscillation can persist under constant conditions of light and temperature. Therefore, it has been accepted by most investigators that the generation mechanism of the circadian rhythm is endogeneous. However, one cannot reject the possibility that these rhythms are caused by some geophysical exogeneous factor having a 24-hour period, such as atmospheric pressure, gravity, or electromagnetic radiation. We use Neurospora crassa band A mutual which shows an obvious circadian rhythm in its spore-forming (conidiation) on the ground, and we intend to attempt the conidation of this mutant in the Spacelab where 24-hour periodicity is severely attenuated and to elucidate the effect of the geophysical exogeneous factor in the generation mechanism of the circadian rhythm.

Miyoshi, Yashuhiro↗

DOE OSTI2024

Engineered living materials for capture, conversion, and recycling technologies

Continued dependence on crude oil and natural gas resources for fossil fuels has caused global atmospheric carbon dioxide (CO 2 ) emissions to increase to record-setting proportions. There is an urgent need for efficient and inexpensive carbon sequestration systems to mitigate large-scale CO 2 emissions from industrial flue gas. Carbonic anhydrase (CA) has shown high potential for enhanced CO 2 capture applications compared to conventional absorption-based methods currently utilized in various industrial settings. This study aims to understand structural aspects that contribute to the stability of CA enzymes critical for their applications in industrial processes, which require the ability to withstand conditions different from their native environments. Here, we evaluated the thermostability and enzyme activity of mesophilic and thermophilic CA variants at different temperature conditions and in the presence of atmospheric gas pollutants like nitrogen oxides (NO x ) and sulphur oxides (SO x ). Based on our enzyme activity assays and molecular dynamics simulations, we see increased conformational stability and CA activity levels in thermostable CA variants incubated week-long at different temperature conditions. The thermostable CA variants also retained high levels of CA activity despite changes in solution pH due to increasing NO x and SO x concentrations. Furthermore, a loss of CA activity was observed only at high concentrations of NO x /SO x that possibly can be minimized with appropriate buffered solutions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

SPRUCE Sphagnum Phytobiome Responses to Whole Ecosystem Warming and Elevated Atmospheric CO2 in July, 2017-2021

This dataset reports the carbon (C) and nitrogen (N) isotopic composition of Sphagnum moss tissue collected from the Spruce and Peatland Responses Under Changing Environments (SPRUCE) experimental site within the Marcell Experimental Forest in northern Minnesota, USA. Tissues were collected inside the SPRUCE experimental study plot, where air and peat warming are combined in whole-ecosystem warming treatments, from 2019-2021. Additionally, rates of nitrogen fixation and methane oxidation measured for Sphagnum from 2017, 2019, and 2021. Sphagnum tissue % water content was measured at all timepoints. This dataset was used to conduct analyses within Petro et al (2023). Sphagnum C and N concentrations and 13C and 15N isotopic natural abundance were determined for three replicate Sphagnum tissue samples collected from inside the SPRUCE enclosures in July 2019-2021. Elemental and isotope analyses were conducted at the University of Georgia – Center for Applied Isotope Studies (CAIS; https://cais.uga.edu/). Plant elemental analysis was performed by the micro-Dumas method, while isotopic natural abundance was measured by isotope ratio mass spectrometry. 13C natural abundance is expressed as the per mille (‰) deviation from the Pee Dee Belemnite standard (PDB) 13C:12C ratio (δ13C), while 15N natural abundance is expressed as the ‰ deviation from the N2 atmospheric 15N:14N ratio (δ15N). Rates of nitrogen fixation and methane oxidation were measured using serum bottle incubations performed with Sphagnum collected inside the SPRUCE enclosures in July 2017, 2019, and 2021. Rates were calculated according to the amount of 15N-N2 or 13C-CH4 (methane) incorporated into the Sphagnum tissue or incubation headspace over the 48-hour intion period. In 2017, incubations were performed in growth chambers set to the temperature of the experimental plots during Sphagnum sampling. In 2019 and 2021, the incubations were performed directly inside the experimental plots. The 13C and 15N isotopic natural abundance data and rate measurements presented in this dataset provide insight into the impacts of whole-ecosystem warming and elevated atmospheric carbon dioxide (CO2) on N and C cycling within the Sphagnum phytobiome. This dataset contains two data files in comma-separate values (*.csv) format. Additional metadata are provided: two data dictionaries and a file-level metadata file in comma-separate values (.csv) format and a user guide in PDF (*.pdf) format. Additional datasets on NH4-N availability, porewater concentrations of CH4 and CO2, and Sphagnum groundcover within the SPRUCE experimental plots are available. These are outlined in the Related Datasets section in the user guide.

delta 13 carbon↗

DOE OSTI2023

Changing temperature profiles and the risk of dengue outbreaks

As temperatures change worldwide, the pattern and competency of disease vectors will change, altering the global distribution of both the burden of infectious disease and the risk of the emergence of those diseases into new regions. To evaluate the risk of potential summer dengue outbreaks triggered by infected travelers under various climate scenarios, we develop an SEIR-type model, run numerical simulations, and conduct sensitivity analyses under a range of temperature profiles. Our model extends existing theoretical frameworks for studying dengue dynamics by introducing temperature dependence of two key parameters: the mosquito extrinsic incubation period and the lifespan of mosquitoes, which empirical data suggests are both highly temperature dependent. We find that changing temperature significantly alters dengue risk in an inverted U-shape, with temperatures in the range 27-31°C producing the highest risk. As temperatures increase beyond 31°C, the determinants of dengue risk begin to shift from mosquito biting rate and carrying capacity to the duration of the human infectious period, suggesting that changing temperatures not only alter dengue risk but also the potential efficacy of control measures. To illustrate the role of spatial and temporal temperature heterogeneity, we select five US cities where the primary dengue vector, the mosquito Aedes aegypti , has been observed, and which have had dengue cases in the past: Los Angeles, Houston, Miami, Brownsville, and Phoenix. Our analysis suggests that an increase of 3°C leads to an approximate doubling of the risk of dengue in Los Angeles and Houston, but a reduction of risk in Miami, Brownsville, and Phoenix due to extreme heat.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2002

Worms on the International Space Station

C. elegans was proposed as a model system for space biology studies in 1991 and has since flown on STS-42, STS-76, and STS-95. Data obtained from these flights have confirmed that C. elegans requires adequate in flight oxygenation and displays an increased rate of mutation, much like other organisms in space. Unlike vertebrates, C. elegans has been observed to successfully complete two continuous full life cycles in space without gross developmental abnormalities. These observations, could with the utility of C. elegans as a terrestrial, fully sequenced, model system make C. elegans a good candidate for long term research onboard the International Space Station (ISS). We are currently working on technology to support biological studies aboard the ISS. A component of this effort is in the development of the Space Station Biological Research Program (SSBRP) Incubator which will be able to house organisms at a constant temperature setpoint ranging from 4deg C to 45deg C. The SSBRP Incubator provides air exchange, power, data and video ports and, when placed in the collaborative NASA/NASDA 2.5M centrifuge rotor, will be capable of providing a lg gravity control. Current plans for validation of the Incubator include video monitoring and periodic sampling of C. elegans in the Incubator onboard the ISS. Once returned to earth, samples will be distributed for analysis via a specimen sharing plan and analyzed for gene expression and other parameters of growth and development in space flight. These data should provide the C. elegans research community with a baseline from which to propose studies for future flights. We have also been developing an appropriate method of culturing C. elegans in liquid media in order to remove the need for the crew to assure that strains are properly fed. Currently, we are growing strains in the chemically defined, axenic, media developed by Dr. Nancy Lu. Wild-type animals complete multiple generations and appear generally healthy after being grown in unchanged media for a minimum of three to four weeks. To assure proper oxygenation we have chosen to grow animals in ten milliliter OptiCells(TradeMark). The choice of the OptiCell(TradeMark) also allows for automation of culturing as demonstrated for cell cultures grown in the OptiCell(TradeMark). Arrangements have been made for the liquid media to be commercially produced by Mediatech, Inc. and OptiCells(TradeMark) are available from BioCrystal Ltd.

Szewczyk, Nate↗