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DOE OSTI2022

Illuminating the pathways to carbon liberation: a systems approach to characterizing the consequential unknowns of carbon transformation and loss from thawing permafrost peatlands (Final Report)

The IsoGenie3 Project delivered new systems-level insights into carbon cycling in thawing permafrost landscapes, with an emphasis on methane and carbon dioxide emissions. From >200 samples from the site collected over a decade, co-analyzed for geochemistry and microbiology, the team recovered ~1,500 assembled microbial genomes and ~1,900 viral population genomes, revealing appreciable genetic novelty - from a new highly abundant bacterial phylum, to novel methane consumers and their activities, to rampant viral novelty. IsoGenie3 linked these organisms to carbon compound transformations (which define the cycling of organic matter in soils, and the loss of the greenhouse gases carbon dioxide and methane), and saw that the microbes at each stage of permafrost thaw had different genetic potential to degrade categories of compounds, expressed that genetic potential differently, and actually transformed carbon compounds into greenhouse gases in different ways. IsoGenie 3 identified that some of the thaw-stage differences were due to plant-microbiome relationships; the plant species across the thaw gradient contributed different carbon compounds into the soil, and hosted distinct microbiota (differing among parts of plants as well as species). Lastly, microbes in the saturated post-thaw conditions appeared likely to contribute to the mobilization and toxification of mercury released during thaw. In parallel with ongoing field sampling and analysis, hypotheses arising from field observations were tested via lab incubation experiments. When communities are taken out of their native habitats, they behave differently, and the team first rigorously quantified the magnitude of this effect on microbiome composition and functional capacity, organic matter composition, and gas production; overall the main system processes were maintained in the lab incubations under the conditions tested. Further, the microbial data could inform geochemical reaction network models of those processes. Then, the team ran experiments with additions of compounds, varying temperature, and “live” vs. “dead” peat (the latter having been gamma irradiated, with a few additional variants to control for methodological artifacts). From these, we (a) determined the importance of plant-derived soluble phenolic compounds in bogs’ extraordinary recalcitrance of organic matter, and carbon gas emissions skewed to carbon dioxide; (b) proposed an abiotic ‘tanning’ mechanism, which could contribute to Sphagnum’s inhibitory effect on anaerobic decomposition through alteration of N availability. IsoGenie3 illuminated longer-term and landscape-scale interactions of permafrost thaw and carbon cycling, advancing knowledge of the drivers of methane dynamics not only across in the permafrost-associated peatland (where hydrology and plant communities dictate microbiomes) but also their interconnected lakes (where sediment carbon quality and resident microbiota are determined by position within lake, and lake features). By leveraging observations of site methane dynamics extending well before this project, the team was able to construct a 44-year portrait of the interplay of permafrost thaw, hydrology, vegetation dynamics, and carbon gas emissions, and the doubling of the fully-thawed fens over this time. From the detailed study of this focal site, IsoGenie3 also aimed to improve model representation of these kinds of sites and processes. To improve predictions of methane transformations, we incorporated acetate and isotope dynamics into the ‘DNDC’ biogeochemistry model. In addition, recovered genomes were grouped into ‘functional groups’, i.e. the genomes that perform a specific function of interest, then used to parameterize maximum growth rate and optimum growth temperature (via signatures in their sequence composition) for the BioCrunch model. The BioCrunch model was then in turn used to test the impact of increasing functional resolution of the microbes, on the carbon gas emissions. Lastly for modeling, the ecosys model was parameterized from the microbial and other data, and used to evaluate drivers of e.g. change in methane emissions. Finally, this project also led to the development of a range of new methods and tools, a new metric of organic matter decomposability, as well as a graph-database solution to multidisciplinary data storage and querying. This project’s ongoing analyses at our focal site also contributed to broader advancements in understanding elements of genetic plasticity and methane metabolism, climate change microbiology and community assembly, global peatland geochemistry and Arctic lakes’ roles in climate feedbacks.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1992

UPT scenarios: Implications for system reliability

The objective of this project was to examine the corrosion resistance of 316L stainless steel in several urine pre-treat solutions. Four solutions were examined: untreated urine (control); urine pretreated with oxone (potassium peroxymonosulfate sulfate); urine pretreated with sodium hypochlorite (NaOCl); and urine pretreated with ozone (O3). In accordance with current procedures, all solutions but the control were acidified to a pH of 2.5 using sulfuric acid--this suppresses the generation of ammonia in the solutions and is intended to limit microbial growth. Welded and unwelded coupons were exposed to each solution. In addition, Titanium coupons (welded and unwelded) were exposed to biologically active environmental control and life support system (ECLSS) water. Microbial attachment and biofilm growth were monitored. Ozone was examined as a biocide/oxidizer/corrosion preventative (simultaneous addition) and as a remediation method (added one week after exposure). In an unrelated effort, HP 9-4-30 coupons were exposed to biologically active solutions. Corrosion rates for welded and unwelded samples were determined--results were correlated to the ongoing HP 9-4-30 weldment stress corrosion study.

Walsh, Daniel W.↗

NASA NTRS2009

Comparative Packaging Study

Future long duration manned space flights beyond low earth orbit will require the food system to remain safe, acceptable and nutritious. Development of high barrier food packaging will enable this requirement by preventing the ingress and egress of gases and moisture. New high barrier food packaging materials have been identified through a trade study. Practical application of this packaging material within a shelf life test will allow for better determination of whether this material will allow the food system to meet given requirements after the package has undergone processing. The reason to conduct shelf life testing, using a variety of packaging materials, stems from the need to preserve food used for mission durations of several years. Chemical reactions that take place during longer durations may decrease food quality to a point where crew physical or psychological well-being is compromised. This can result in a reduction or loss of mission success. The rate of chemical reactions, including oxidative rancidity and staling, can be controlled by limiting the reactants, reducing the amount of energy available to drive the reaction, and minimizing the amount of water available. Water not only acts as a media for microbial growth, but also as a reactant and means by which two reactants may come into contact with each other. The objective of this study is to evaluate three packaging materials for potential use in long duration space exploration missions.

Perchonok, Michele H.↗

DOE OSTI2021

Trade-Offs Between Growth Rate and Other Fungal Traits

If we better understand how fungal responses to global change are governed by their traits, we can improve predictions of fungal community composition and ecosystem function. Specifically, we can examine trade-offs among traits, in which the allocation of finite resources toward one trait reduces the investment in others. We hypothesized that trade-offs among fungal traits relating to rapid growth, resource capture, and stress tolerance sort fungal species into discrete life history strategies. We used the Biolog Filamentous Fungi database to calculate maximum growth rates of 37 fungal species and then compared them to their functional traits from the fun fun database. In partial support of our hypothesis, maximum growth rate displayed a negative relationship with traits related to resource capture. Moreover, maximum growth rate displayed a positive relationship with amino acid permease, forming a putative Fast Growth life history strategy. A second putative life history strategy is characterized by a positive relationship between extracellular enzymes, including cellobiohydrolase 6, cellobiohydrolase 7, crystalline cellulase AA9, and lignin peroxidase. These extracellular enzymes were negatively related to chitosanase 8, an enzyme that can break down a derivative of chitin. Chitosanase 8 displayed a positive relationship with many traits that were hypothesized to cluster separately, forming a putative Blended life history strategy characterized by certain resource capture, fast growth, and stress tolerance traits. These trait relationships complement previously explored microbial trait frameworks, such as the Competitor-Stress Tolerator-Ruderal and the Yield-Resource Acquisition-Stress Tolerance schemes.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRSDate not supplied

Microbial Monitoring of New Cleanrooms Used to Curate Astrobiologically Relevant Asteroid Samples from Bennu and Ryugu

Introduction: NASA has constructed two new cleanrooms to house materials from the OSRIS-REx and Hayabusa2 missions to the asteroids Ryugu (162173) and Bennu (101955), respectively. In accordance with standard astromaterials curation practices, these cleanrooms will be monitored for particulate contamination and maintained to ISO 5 equivalent standards1. Since the samples in these collections are expected to contain prebiotic organic compounds that may help explain the origin of life on Earth, these labs will also be monitored for organic and biological contamination2. Samples from Ryugu arrived on Earth in December, 2020. After basic characterization in Japan, NASA received a subset of these samples at the astromaterials curation facility in Houston in December of 2021. OSIRIS-REx is expected to return samples in September, 2023. Here we present preliminary microbial monitoring results from monthly monitoring of these new labs and the connected microtomy and staging areas that support them, as they are being commissioned. We also compare these results to baseline values for other astromaterials curation labs. We will also briefly describe additional cleaning efforts employed to reduce the bioburden in these new cleanrooms. Methods: Microbial samples were collected from surfaces using a dry macrofoam swab (Puritan Brand 2518051PFRNDFD). Swabs were also opened in the lab but not touched to any surfaces to function as negative controls. Samples and controls were processed inside a class II biosafety cabinet to avoid inadvertent cross contamination. The swabs were suspended in 15 ml of PBS (Phosphate Buffered Saline) and vortexed for 20 seconds to remove cells from the swab surface. The PBS was used to inoculate Petri dishes filled with TSA (Tryptic Soy Agar), Blood Agar, or Reasoners 2 agar to check for microbial growth. Each plate was inoculated with 0.1 ml of PBS. The TSA and blood agar plates were incubated at 35˚C and the Reasoners 2 agar plates were incubated at 25˚C for seven days. Petri dishes filled with Potato dextrose agar, Saboraud dextrose agar, or Saboraud dextrose agar with 0.1 mg/ml of chloramphenicol, an antibiotic, were used to check for fungal growth. These plates were inoculated with 0.3 ml of PBS and incubated at 30˚C. The remaining PBS was frozen at -80 ˚C for DNA sequencing. After incubation, isolates were counted and reisolated for identification. Isolates were identified using the VITEK23 system or by sequencing a portion of the 16S rRNA gene for bacteria or the ribosomal internal transcribed spacer (ITS) for fungi. Sequencing was performed with an ABI 3500 Sanger sequencer. Results: During our initial sampling, six of the seven sites sampled (86%) displayed bacterial or fungal growth. Samples collected from the staging areas and microtomy labs are not included in this calculation since those areas are maintained at a lower ISO 7 equivalent cleanliness standard. A month later, only three of the seven sites (43%) displayed bacterial growth. No fungal growth was detected in the second sampling. Since new equipment had been introduced to the Hayabusa2 lab since the first round of sampling, an additional three sampling sites were included in the second round of sampling. None of these sites displayed microbial growth. These sites will be included in all future sampling efforts. Bacterial isolates have been identified from the following genera at multiple time points: Micrococcus, Staphylococcus, and Bacillus. Isolates from the genera: Microbacterium, Nocardioides, Methylocystis, and Microvirga were identified in the initial sampling, but were not present at later time points. Identification of fungal isolates is in progress. Results are summarized in Table 1. Discussion: The recovery rate or percentage of positive samples4 was initially 86%, which is higher than the median recovery rate for comparable ISO 5 equivalent curation labs like Stardust (33%), Hayabusa (33%), and Cosmic Dust (50%). However, after a month of operation, the recovery rate for these same sites decreased to 43%, which is similar to what we observe in comparable curation cleanrooms with no microbial control requirements. Adding in the new sampling sites further decreases the recovery rate to 30%. With the reduction in recovery rate, we also observed a decrease in microbial diversity. At the first time point, we observed at least 10 different bacterial species and at least two different fungi. This is a higher diversity than the median values for comparable ISO 5 equivalent labs (2-4 isolates per sampling event). After the second sampling, we observed at least 4 bacterial species and no fungi, which is more consistent with comparable labs. We expect the recovery rate and diversity in both labs to continue to decrease as routine operation continues. We will use ultrapure hydrogen peroxide to disinfect equipment and work areas prior to opening any sample containers. Most of the bacterial and fungal isolates were detected on samples from the cleanroom floors. This is consistent with baseline results from other curation labs. Organisms from the genera Bacillus, Staphylococcus, and Micrococcus that were repeatedly detected are common in cleanrooms and on human skin5,6. These organisms are generally thought to be introduced when people enter the cleanroom. Microbacterium, Nocardioides, and Microvirga have also previously been identified in astromaterials cleanrooms, but not as frequently as Bacillus, Staphylococcus, and Micrococcus. Methylocystis is a novel genus in the astromaterials cleanrooms, but it was identified with low accuracy (93% match in the sequenced region of the 16S rRNA gene) and further work is needed to confirm this identification. Microbacterium is a diverse genus with isolates identified from terrestrial and aquatic sediments. Some species of Microbacterium are capable of degrading complex organic compounds found in crude oil. The presence of these bacteria in the OSIRIS REx and Hayabusa2 cleanrooms should be closely monitored. Methylocystis is a genus of methanotrophic bacteria capable of oxidizing methane. If this identification proves to be correct and it is detected again, it should be closely monitored as well. Under nominal operating conditions, samples should not ever encounter the cleanroom floor or other high traffic areas. If we observe an increase in the bioburden in sensitive work areas that appears to be influenced by organism transfer from high traffic areas like the floors, we can employ additional hydrogen peroxide treatments to disinfect high traffic areas. Routine microbial monitoring of these labs will ensure that NASA’s astromaterials collections remain pristine and useful for scientific study. Table 1. Sampling Locations and Colony Counts Bacterial CFUa Fungal CFU Bacterial CFU Fungal CFU Lab - Location 11/2/2021 11/2/2021 12/13/2021 12/13/2021 H2b-Floor 4 8 1 0 H2-staging pass through 3 0 0 0 H2-microtomy pass through TNTCc 0 0 0 H2 Microscope 1 NA NA 0 0 H2 Microscope 2 NA NA 0 0 H2-Table NA NA 0 0 OREXd- microtomy pass through 0 0 6 0 OREX – Anteroom pass through 0 0 0 0 OREX – Floor 1 2 0 0 OREX Witness Foil Table 3 0 1 0 Staging-Floor 16 0 15 0 Microtomy-Floor 3 0 2 0 a: CFU = Colony Forming Unit b: H2 = Hayabusa2 Lab c: TNTC = too numerous to count d: OREX = OSIRIS-REx Lab References: 1. ISO 14644-1:2015 - Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration. 37 (2015). 2. McCubbin, F. M. et al. Space Sci Rev 215, (2019). 3. Pincus, D. H. Encyclopedia of Rapid Microbiological Methods (2005). 4. The United States Pharmacopeial Convention. USP General Chapter <1116> 17, 784–794 (2013). 5. Sheraba, N. S., Yassin, A. S. & Amin, M. BMC Research Notes 3, 278 (2010). 6. Utescher, C. L. de A., Franzolin, M. R., Trabulsi, L. R. & Gambale, V. Brazilian Journal of Microbiology 38, 710–716 (2007).

A B Regberg↗

NASA NTRS1986

Biological methanogenesis and the CO2 greenhouse effect

It is well established that plants tend to increase net photosynthesis under increased carbon dioxide. It is also well established that a large fraction of atmospheric methane is produced by microbial metabolism of organic sediments in paddies and freshwater wetlands, where a major source of organic debris is local plant growth. As CO2 increases, it may lead to increased methane production and a resulting enhancement of the expected greenhouse warming. A rough estimate of the present rate of this biologically mediated feedback on the climate system indicates that it might account for as much as 30 percent of the observed methane increase and speed up the greenhouse forcing by as much as 15 percent.

Guthrie, P. D.↗

DOE OSTI2023

Cell Free and Immobilization Technologies (CFIT)

Today, several key factors negatively impact the production of fuels and chemicals from renewable sources. Common hindrances in the biological production of biochemicals are: (1) end-product or intermediate toxicity to the microbial biocatalyst, (2) the diversion of carbon to biomass formation, and (3) co-production of undesired byproducts. A particularly attractive alternative is to eliminate the biocatalyst entirely and instead operate the desired metabolic pathways in isolation, thus circumventing the roadblocks of biological toxicity, lower yields, and lack of specificity. However, cell-free enzyme systems still suffer from low productivities owing in part to the effects of free diffusion of intermediates, lack of long term enzyme stability, cofactor cost or inefficient recycling rates, and finally, the cost of enzyme production/purification. This project represents a new effort to propose innovative and cost competitive routes to producing biochemicals from a variety of feedstocks using cell free approaches. These routes will help reduce the current risk and cost associated with classical cell free production. Cell free technologies show promise for application to the production of toxic/inhibitory products or products difficult to separate from microbial growth media and can help reduce the production barriers in multiple areas of biological conversion of feedstocks to biochemicals. More specifically, we are developing new technologies and routes that could be used to produce high value biochemicals such as 2,3 BDO and terpenes (among many others) from biomass derived C5/C6 sugars or lignin but also from waste byproducts such as glycerol. This project will lead to significant innovation and also lead to new concepts and rational design of pathways and enzymes. Within this project, we will develop new metabolic enzyme cascades that will represent natural or artificial combinations of enzymes to produce the desired biochemicals from a variety of feedstocks. We are also developing basic design principals for constructing synthetic metabolons, using fusion proteins and synthetic protein scaffolds, to promote substrate channeling and stability while conserving peak activity. Additionally, our efforts include a techno economic analysis (TEA) of cell free approaches to provide the sensitivities of the process to enzyme loading, activity, pH, reactor volumes, cofactor recycling rates. Finally, we are focusing on further increasing stability, operating lifetime and efficiency of the pathway enzymes by immobilization on support surfaces. We are also focus on immobilizing pathway enzymes or combinations of enzymes on several different conducting polymers and evaluate the effect on stability and operating lifetime. As more combinations become available we will conduct a more systematic study of the means of immobilizing these enzymes. This preliminary work will enable the in-depth study of cofactor recycling at these interfaces using mediators for electron transfer. Taken together, these approaches will enable process intensification, continuous operation, lower capital and separations costs and end-product flexibility, and thus has the potential to contribute significantly to BETO's goals of cost competitive biofuels and bioproducts. To date we have demonstrated 1) the conversion of pyruvate to 2,3 BDO (4 enzymes with cofactor recycling, no additional cofactors needed) at >100g/L (>3g/L/h) using our enzyme tethering approach, 2) Generated mutants of a key redox enzyme with >180- fold improvement in NADH utilization over WT to >85% of NADPH utilization. 3) Produced mevalonate at >10g/L from glucose, limonene at >10g/L from mevalonate and more than >5g/L from glucose with crude enzyme preparations and complete cofactor recycling. 4) successfully generated fully active cross-linked aggregates (CLEAs) of NOX, a cofactor-regenerating water-forming oxidase that is key to our process with increased stability 5) Engineered and identified enzymes able to use the biomimetic cofactor NMN and synthesized new synthetic cofactors.

biocatalysis↗

DOE OSTI2026

Data for Impacts of Legacy and Contemporary Nitrogen Inputs on N2O and CO2 Emissions in Miscanthus and Maize Cultivated Soils

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 × giganteu s ( 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 M × g 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.

Carbon↗

DOE OSTI2021

Lipid production from non-sugar compounds in pretreated lignocellulose hydrolysates by Rhodococcus jostii RHA1

Valorization of all carbon sources from biorefinery wastes to biofuels and bioproducts is an attractive but challenging strategy to improve the carbon efficiency of the entire biorefinery process for economic competitiveness. Rhodococcus jostii RHA1 is considered as a promising candidate for conversion of non-sugar compounds in pretreated lignocellulose hydrolysate, which contain aromatic monomers, furans and acids derived from biomass. However, the insufficient understanding of the strain’s complex metabolism network limits its application. In this study, furans (furfural, 5-hydroxymethylfurfural), phenols (vanillin, vanillate), and organic acids (acetic acid), which mimic the complex components of the non-sugar compounds in pretreated biomass hydrolysate, were mixed with benzoate or used individually as carbon sources to investigate the growth and lipid accumulation of R. jostii RHA1. Fermentation kinetics, specific growth rates (h-1) and lipid yield (g(g) -1, g(mmol)-1), for each selected biomass-derived compound were compared for lipid production. The order of the specific growth rates (h-1) was benzoate > acetic acid > vanillate > vanillin. Higher consumption rate of benzoate than that of vanillate, as well as different lipid yield from them, suggested the preference of strain employing two branches of ß-ketoadipate pathway and plausibly distinct carbon routing to lipid biosynthesis. And acetic acid showed the lowest lipid yield (g(mmol)-1) indicating less carbon contributed to lipid production. The highest lipid content (0.46 g lipid/g CDW) was achieved using benzoate as a main carbon source in the presence of 5-hydroxymethylfurfural (5-HMF). The generated lipids predominantly contained C16:0 and C17:0 fatty acids. The concentration of furfural and 5-HMF in fermentation broth decreased individually or simultaneously with benzoate catabolism. NMR results revealed the oxidation of furfural and 5-HMF by R. jostii RHA1. This study provides new insights into the catabolism and metabolism of non-sugar compounds derived from biomass by Rhodococcus in terms of growth and lipid production, facilitating its application to 2nd generation biofuel production.

Li, Xiaolu↗

DOE OSTI2022

Coupling gas purging with inorganic carbon supply to enhance biohydrogen production with Clostridium thermocellum

Clostridium thermocellum is a desirable biocatalyst for biohydrogen production, with a native ability to simultaneously saccharify cellulose and to metabolize released cellodextrins for hydrogen production. During fermentation with C. thermocellum , partial pressures of two gases - CO 2 and H 2 - are critical drivers of overall reaction kinetics. Biohydrogen production is enhanced by maintaining a low hydrogen partial pressure, while high concentrations of dissolved CO 2 promote microbial biomass synthesis. Our study evaluates the inherent trade-offs between hydrogen stripping and inorganic carbon supply for optimized biohydrogen synthesis. Here, we find that nitrogen sparging at low flow rates increases hydrogen production when compared with an equivalent nitrogen overlay, but that high rates of nitrogen sparging inhibit cell growth and hydrogen production. Decreasing dissolved hydrogen partial pressure via nitrogen sparging also lowers the production of reduced metabolites, including lactate and ethanol. To address potential stripping of inorganic carbon from the production medium, we supplemented CO 2 to the sparging gas and co-optimized for gas flow rate and for the CO 2 fraction of the sparging gas. Total hydrogen production increased from 50 mmol∙L -1 in the base condition, when the bioreactor was sparged with 0.1 LPM of pure nitrogen, to 181.3 mmol∙L -1 when sparged with 1.3 LPM of 33 % CO 2 , demonstrating that biohydrogen production is highly sensitive to both parameters. Fine sensitivity of biohydrogen production to sparging conditions highlights the critical importance of bioreactor design and operation to achieve maximum H 2 removal without compromising inorganic carbon supply to bacterial central metabolism.

09 BIOMASS FUELS↗

DOE OSTI2022

Water-soluble saponins accumulate in drought-stressed switchgrass and may inhibit yeast growth during bioethanol production

Abstract Background Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. Results Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae . Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography–mass spectrometry (LC–MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. Conclusions Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Electrons to Molecules by Engineering and Evolution: Biological Upgrading of Formate by Cupriavidus necator

Waste carbon from industrial point sources can be captured, stored, and/or transformed using electrochemical conversion or "electrons to molecules" technologies using low-cost renewable electricity. One such process involves electrocatalytic reduction of CO2 to generate formate/formic acid, a C1 carboxylic acid. Formate is a promising potential feedstock for microbial upgrading, as it is water soluble and can be consumed as the sole source of carbon and energy by some microbial species, such as the soil bacterium Cupriavidus necator. Here we will present progress toward improving C. necator as a host for biological conversion of formate to value-added products. Using the power of adaptive laboratory evolution, we were able to isolate mutants of C. necator with significantly faster growth rates on formate. We then sequenced the genomes of these strains, elucidated the metabolic role of the mutations we found, and then used these insights to build rationally engineered strains that outperform even the best evolved isolates. These results highlight the utility of "genome streamlining" as a route for generating platform strains with potential industrial applications.

adaptive laboratory evolution↗

DOE OSTI2022

The survivor strain: isolation and characterization of Phormidium yuhuli AB48, a filamentous phototactic cyanobacterium with biotechnological potential

Despite their recognized potential, current applications of cyanobacteria as microbial cell factories remain in early stages of development. This is partly due to the fact that engineered strains are often difficult to grow at scale. This technical challenge contrasts with the dense and highly productive cyanobacteria populations thriving in many natural environments. It has been proposed that the selection of strains pre-adapted for growth in industrial photobioreactors could enable more productive cultivation outcomes. Here, we described the initial morphological, physiological, and genomic characterization of Phormidium yuhuli AB48 isolated from an industrial photobioreactor environment. P. yuhuli AB48 is a filamentous phototactic cyanobacterium with a growth rate comparable to Synechocystis sp . PCC 6803. The isolate forms dense biofilms under high salinity and alkaline conditions and manifests a similar nutrient profile to Arthrospira platensis ( Spirulina ). We sequenced, assembled, and analyzed the P. yuhuli AB48 genome, the first closed circular isolate reference genome for a member of the Phormidium genus. We then used cultivation experiments in combination with proteomics and metabolomics to investigate growth characteristics and phenotypes related to industrial scale cultivation, including nitrogen and carbon utilization, salinity, and pH acclimation, as well as antibiotic resistance. These analyses provide insight into the biological mechanisms behind the desirable growth properties manifested by P. yuhuli AB48 and position it as a promising microbial cell factory for industrial-scale bioproduction[221, 1631].

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H 2 :CO feedstock ratios for enhancing carbon capture efficiency

Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO 2 ) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H 2 :CO to maximize CO 2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO 2 , we established controlled chemostats that facilitated a novel and high (11:1) H 2 :CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H 2 :CO condition where ~ 50% of the carbon in ethanol is derived from CO 2 . Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H 2 :CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H 2 :CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

09 BIOMASS FUELS↗

NASA NTRS1995

Urine Pretreat Injection System

A new method of introducing the OXONE (Registered Trademark) Monopersulfate Compound for urine pretreat into a two-phase urine/air flow stream has been successfully tested and evaluated. The feasibility of this innovative method has been established for purposes of providing a simple, convenient, and safe method of handling a chemical pretreat required for urine processing in a microgravity space environment. Also, the Oxone portion of the urine pretreat has demonstrated the following advantages during real time collection of 750 pounds of urine in a Space Station design two-phase urine Fan/Separator: Eliminated urine precipitate buildup on internal hardware and plumbing; Minimized odor from collected urine; and Virtually eliminated airborne bacteria. The urine pretreat, as presently defined for the Space Station program for proper downstream processing of urine, is a two-part chemical treatment of 5.0 grams of Oxone and 2.3 ml of H2SO4 per liter of urine. This study program and test demonstrated only the addition of the proper ratio of Oxone into the urine collection system upstream of the Fan/Separator. This program was divided into the following three major tasks: (1) A trade study, to define and recommend the type of Oxone injection method to pursue further; (2) The design and fabrication of the selected method; and (3) A test program using high fidelity hardware and fresh urine to demonstrate the method feasibility. The trade study was conducted which included defining several methods for injecting Oxone in different forms into a urine system. Oxone was considered in a liquid, solid, paste and powered form. The trade study and the resulting recommendation were presented at a trade study review held at Hamilton Standard on 24-25 October 94. An agreement was reached at the meeting to continue the solid tablet in a bag concept which included a series of tablets suspended in the urine/air flow stream. These Oxone tablets would slowly dissolve at a controlled rate providing the proper concentration in the collected urine. To implement the solid tablet in a bag approach, a design concept was completed with prototype drawings of the complete urine pretreat prefilter assembly. A successful fabrication technique was developed for retaining the Oxone tablets in a fabric casing attached to the end of the existing Space Station Waste Collection System urine prefilter assembly. The final pretreat prefilter configuration held sufficient Oxone in a tablet form to allow normal scheduled daily (or twice daily) change out of the urine filter depending on the use rate of the Space Station urine collection system. The actual tests to prove the concept were conducted using the Urine Fan/Separator assembly that was originally used in the STS-52 Design Test Objective (DTO) urinal assembly. Other related tests were conducted to demonstrate the actual minimum ratio of Oxone to urine that will control microbial growth.

Source record↗

NASA NTRS2003

Development of the Next Generation Gas Trap for the Space Station Internal Thermal Control System

The current dual-membrane gas trap is designed to remove non-condensed gases (NCG) from the Internal Thermal Control System (ITCS) coolant on board the International Space Station (ISS). To date it has successfully served its purpose of preventing depriming, overspeed, and shutdown of the ITCS pump. However, contamination in the ITCS coolant has adversely affected the gas venting rate and lifetime of the gas trap, warranting a development effort for a next-generation gas trap. Design goals are to meet or exceed the current requirements to (1) include greater operating ranges and conditions, (2) eliminate reliance on the current hydrophilic tube fabrication process, and (3) increase operational life and tolerance to particulate and microbial growth fouling. In addition, the next generation gas trap will essentially be a 'dropin" design such that no modifications to the ITCS pump package assembly (PPA) will be required, and the implementation of the new design will not affect changes to the ITCS operational conditions, interfaces, or software. This paper will present the initial membrane module design and development work which has included (1) a trade study among several conceptual designs, (2) performance modeling of a hydrophobic-only design, and (3) small-scale development test data for the hydrophobic-only design. Testing has shown that the hydrophobic-only design is capable of performing even better than the current dual-membrane design for both steady-state gas removal and gas slug removal.

Leimkuehler, Thomas O.↗

DOE OSTI2024

Fungal community composition predicts forest carbon storage at a continental scale

Forest soils harbor hyper-diverse microbial communities which fundamentally regulate carbon and nutrient cycling across the globe. Directly testing hypotheses on how microbiome diversity is linked to forest carbon storage has been difficult, due to a lack of paired data on microbiome diversity and in situ observations of forest carbon accumulation and storage. Here, we investigated the relationship between soil microbiomes and forest carbon across 238 forest inventory plots spanning 15 European countries. We show that the composition and diversity of fungal, but not bacterial, species is tightly coupled to both forest biotic conditions and a seven-fold variation in tree growth rates and biomass carbon stocks when controlling for the effects of dominant tree type, climate, and other environmental factors. This linkage is particularly strong for symbiotic endophytic and ectomycorrhizal fungi known to directly facilitate tree growth. Since tree growth rates in this system are closely and positively correlated with belowground soil carbon stocks, we conclude that fungal composition is a strong predictor of overall forest carbon storage across the European continent.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Microbe-iron interactions control lignin decomposition in soil

Lignin decomposition is critically linked to terrestrial carbon (C) cycle due to the enormous C mass of lignin and its importance in controlling overall rates of litter decomposition. Interactions between lignin and iron (Fe) minerals have been increasingly recognized as key mediators of lignin decomposition in experimental studies. However, we still lack a quantitative understanding of how Fe minerals interact with microbes to control lignin decomposition. Here, we leveraged experimental results from an incubation of Fe-rich soil, in which lignin decomposition rates were measured at aerobic conditions after four levels of pre-treated O2 availability, to examine microbe-Fe (MiFe) interactions in lignin decomposition with a MiFe model. We quantified how Fe redox cycling interacted with microbial activities to control lignin decomposition via data-model integration. Our results showed that the MiFe model with time-dependent growth and mortality functions better represented CO2 release from lignin decomposition (R2 ranging from 0.96 to 0.97) than models assuming either first-order or Michaelis-Menten kinetics. Reduction of Fe(III) to Fe(II) after pre-treatments with lower O2 availability stimu-lated the Fenton reaction to break down macro-molecular lignin into small molecules available to microbes. The small molecules of lignin and necromass bounded with oxidized Fe and were protected from decomposition. After 1-year incubation, the model implied that most of C stabilized with Fe minerals was derived from small molecular lignin C. Our quantitative analysis of microbe-Fe interactions sheds new light on lignin decomposition and preservation and helps improve model prediction of soil C persistence under global change.

Liao, Cuijuan↗