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At least 73 records · Page 4

An integrated process for butanol production from cellulosic biomass and CO 2 using engineered clostridia in a linear immobilized bioreactor

Engineered platforms using mixed microbial consortia to consolidate multiple reaction steps and perform disparate conversions simultaneously can substantially enhance the carbon efficiency of biomass conversion processes and lower the biofuel cost to an economically competitive level. In this project, an integrated process will be developed to produce n-butanol from cellulosic biomass and waste gas streams using two engineered clostridial strains, one butanol-tolerant solventogenic strain and one carboxydotrophic acetogen, co-immobilized in a “Linear Immobilized Bioreactor” (LIBR). The CO 2 and H 2 produced by the solventogenic strain will be used by the carboxydotrophic strain to produce acetate (and butyrate) that in turns can be used by the solventogenic strain to produce more butanol. Such a co-cultured fermentation has the potential to increase butanol production from biomass hydrolysate sugars by up to 50% with 100% reduction in CO 2 emission. Butanol produced in the fermentation will be stripped by the rising bubbles of fermentation produced gases (CO 2 and H 2 ) in the LIBR and separated via a condenser, alleviating butanol inhibition while achieving continuous butanol production at a high productivity and product titer, which can reduce both water and energy consumptions by over 50% compared to conventional ethanol and ABE fermentation processes. Phase I study proved the new bioprocess concept and verified our hypothesis that the proposed LIBR and co-cultured fermentation can enhance the conversion of lignocellulosic sugars to butanol with higher butanol yield, productivity and titer, leading to reduced biobutanol cost to compete more favorably with bioethanol and fossil fuels. A prototype lab-scale LIBR for multiphase fermentation was constructed to investigate and demonstrate the technical feasibility and advantages of the LIBR for multiphase fermentation involving sugars and gaseous substrates. The LIBR will be optimized and evaluated for its economic feasibility for biobutanol production from lignocellulosic biomass and industrial waste gases in Phase II.

09 BIOMASS FUELS↗

Syntrophic Co-Cultures of Clostridium Organisms to Produce Higher Alcohols & Other C6-C8 Metabolites (Final Report)

The goal of this project was to advance the systems biology understanding and predictive modeling of synthetic & syntrophic Clostridium microbial consortia, focusing on elucidation of metabolic networks and environmental signals in the consortia. The project has direct applicability to lignocellulosic-biomass based production of higher alcohols as advanced biofuels and C6-C8 metabolites, that can be used as chemicals or serve as biofuel precursors. This project studied and optimized three synthetic syntrophic systems. It examined the population dynamics using flow-cytometry, time-lapse microscopy and PCR analysis. 13C-based tracer analyses was used to examine the metabolite exchange between the syntrophic cell populations and the impact of those interactions on the transcriptome of the individual populations. To enhance our analytical and predictive capabilities, genome-scale models (GSMs) for these syntrophies was developed. RNAseq data for these syntrophic coculture systems were acquired to enable a molecular level understanding of the syntrophies aiming to identify the genetic networks of each organism in the co-culture and compare those against the networks of pure cultures.

09 BIOMASS FUELS↗

Multi-scale Simulation, Calibration, and Optimization of Calcium Carbonate Precipitation in Microbial Communities

Ensuring the efficient engineering of microbially induced calcium carbonate precipitation (MICP) is crucial for a variety of environmental and civil engineering applications, such as soil stabilization and carbon sequestration. Addressing this need, we present a comprehensive multi-scale workflow that begins with the isolation of calcium carbonate-producing microbes from soil samples, followed by metagenomic sequencing and metabolic reconstruction. We then characterize microbial growth phenotypes under diverse nutrient conditions, compare observed growth with metabolic model predictions, and apply the Consistent Reproduction of Phenotype (CROP) algorithm to refine these models. Furthermore, we analyze metabolite consumption and production, and develop a consumer-resource model that is calibrated using time-series measurements of growth rates, pH levels, and calcium carbonate precipitation. The primary benefit of our approach lies in its ability to predict and control MICP outcomes, facilitated by a Bayesian methodology that incorporates priors on initial conditions and parameters. This allows us to compute posteriors by integrating experimental data, and to solve a risk optimization problem under uncertainty to identify nutrient conditions that maximize calcium carbonate production. In contrast to non-Bayesian methods, which fail to quantify uncertainty accurately, our approach provides a more reliable pathway to optimizing nutrient conditions, enhancing the likelihood of achieving desired MICP outcomes. This positions our method as a superior alternative in the quest to improve MICP through engineered microbial consortia.

54 ENVIRONMENTAL SCIENCES↗

Leverage Microbial Innovations to Address Methane Emission Challenges: Input for FY24 Annual LDRD Report

Sandia researchers are addressing the urgent challenge of minimizing dilute and distributed methane emissions. The team is focused on generating stable methane-consuming microbial consortia for deployment in engineered environmental systems. This innovative work aims to produce stable inocula of these consortia and implement viral controls for microbes that generate methane, significantly reducing emissions.

54 ENVIRONMENTAL SCIENCES↗

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↗

Cyanobacteria as cell factories for the photosynthetic production of sucrose

Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds (e.g., polyhydroxybutyrates, 3-hydroxypropionic acid, or dyes) in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential.

59 BASIC BIOLOGICAL SCIENCES↗

Crops for Carbon Farming

Agricultural cropping systems and pasture comprise one third of the world’s arable land and have the potential to draw down a considerable amount of atmospheric CO 2 for storage as soil organic carbon (SOC) and improving the soil carbon budget. An improved soil carbon budget serves the dual purpose of promoting soil health, which supports crop productivity, and constituting a pool from which carbon can be converted to recalcitrant forms for long-term storage as a mitigation measure for global warming. In this perspective, we propose the design of crop ideotypes with the dual functionality of being highly productive for the purposes of food, feed, and fuel, while at the same time being able to facilitate higher contribution to soil carbon and improve the below ground ecology. We advocate a holistic approach of the integrated plant-microbe-soil system and suggest that significant improvements in soil carbon storage can be achieved by a three-pronged approach: (1) design plants with an increased root strength to further allocation of carbon belowground; (2) balance the increase in belowground carbon allocation with increased source strength for enhanced photosynthesis and biomass accumulation; and (3) design soil microbial consortia for increased rhizosphere sink strength and plant growth-promoting (PGP) properties.

54 ENVIRONMENTAL SCIENCES↗

Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

Extremely thermophilic microbial communities associated with the siliceous vent walls and outflow channel of Octopus Spring, Yellowstone National Park, have been examined for lipid biomarker and carbon isotopic signatures. These data were compared with that obtained from representatives of three Aquificales genera. Thermocrinis ruber, Thermocrinis sp. HI, Hydrogenobacter thermophilus, Aquifex pyrophilus and Aquifex aeolicus all contained phospholipids composed not only of the usual ester-linked fatty acids, but also ether-linked alkyl moieties. The fatty acids of all cultured organisms were dominated by very distinct pattern of n-C-20:1 and cy-C-21 compounds. The alkyl glycerol ethers were present primarily as C-18:0 monoethers with the exception of the Aquifex spp. in which dialkyl glycerol ethers with a boarder carbon-number distribution were also present. These Aquificales biomarker lipids were the major constituents in the lipid extracts of the Octopus Spring microbial samples. Two natural samples, a microbial biofilm growing in association with deposition of amorphous silica on the vent walls at 92 C, and the well-known "pink-streamer community" (PSC), siliceous filaments of a microbial consortia growing in the outflow channel at 87 C were analyzed. Both the biofilm and PSC samples contained mono- and dialkyl glycerol ethers with a prevalence of C-18 and C-20 alkyls. Phospholipid fatty acids were comprised of both the characteristic. Additional information is contained in the original extended abstract.

Jahnke, Linda L.↗

Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

Non-NASA Center↗

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria, bacteria-polymer mixtures and biofilms

Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.

NASA Discipline Number 61-10↗

Waste treatment integration in space

The circumstances and criteria for space-based waste treatment bioregenerative life-support systems differ in many ways from those needed in terrestrial applications. In fact, the term "waste" may not even be appropriate in the context of nearly closed, cycling, ecosystems such as those under consideration. Because of these constraints there is a need for innovative approaches to the problem of "materials recycling". Hybrid physico-chemico-biological systems offer advantages over both strictly physico-chemico or biological approaches that would be beneficial to material recycling. To effectively emulate terrestrial cycling, the use of various microbial consortia ("assemblies of interdependent microbes") should be seriously considered for the biological components of such systems. This paper will examine the use of consortia in the context of a hybrid-system for materials recycling in space.

Waste Management/methods↗

Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM—often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)—are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1–9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862–866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. Finally, the morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

54 ENVIRONMENTAL SCIENCES↗

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

59 BASIC BIOLOGICAL SCIENCES↗

CarbStor: Development, Analysis and Modification of Carbon Storing Model Soil Communities

Soil microbial communities carry out a number of key processes including plant growth promotion, bioremediation and cycling of nutrients. Carbon cycling is among the most important of these nutrients that are metabolized and processed by the soil microbial community. Many of the carbon inputs are converted to alternative organic forms of carbon that can be used by plants or act as biomass for microbial growth. However, inorganic forms of carbon can also be produced by soil microbial communities including calcium carbonate (CaCO 3 ). Production of calcium carbonate is beneficial for the ecosystem in several ways: it can stabilize soils and improve soil health, especially denser soils with high clay content, it can act as a method of bioremediation, it can serve as an alternative carbon source for plants and it can be a way to store carbon in soil in a stable, inorganic manner for the long term. While the chemistry surrounding individual species carrying out this process is well known what is lacking is an understanding of how species interact in a community to drive carbonate production. As all microbial species in soil exist in a community setting gaining this knowledge is critical to our predicting and controlling this microbial phenotype to greatly improve soil health. The CarbStor project is focused on developing, analyzing and modifying defined microbial soil consortia that express phenotypes at both the species and community level to convert carbon into recalcitrant stable sources such as precipitated carbonate or microbial necromass. To take full advantage of the soil community for this process we will need to fill several key knowledge gaps (KG), three of which are the focus of CarbStor. KG1: Whether and to what degree microbial communities can be developed that produce precipitated carbon via microbial metabolism. KG2: What interspecies interactions drive the individual member phenotypes in defined communities that lead to carbon precipitation. KG3: How can these interactions be modified to enhance carbon sequestration beyond what native communities are capable of. We hypothesize that in a carbon sequestering community only a subset of species will express phenotypes related to carbon storage processes. We also hypothesize that these phenotypes are expressed as a result of interactions with other species in the community that are not involved in carbon storage processes and that these interactions can be harnessed to enhance community carbon sequestration.

54 ENVIRONMENTAL SCIENCES↗

Soil microbiome interventions for carbon sequestration and climate mitigation

Mitigating climate change in soil ecosystems involves complex plant and microbial processes regulating carbon pools and flows. Here, we advocate for the use of soil microbiome interventions to help increase soil carbon stocks and curb greenhouse gas emissions from managed soils. Direct interventions include the introduction of microbial strains, consortia, phage, and soil transplants, whereas indirect interventions include managing soil conditions or additives to modulate community composition or its activities. Approaches to increase soil carbon stocks using microbially catalyzed processes include increasing carbon inputs from plants, promoting soil organic matter (SOM) formation, and reducing SOM turnover and production of diverse greenhouse gases. Marginal or degraded soils may provide the greatest opportunities for enhancing global soil carbon stocks. Among the many knowledge gaps in this field, crucial gaps include the processes influencing the transformation of plant-derived soil carbon inputs into SOM and the identity of the microbes and microbial activities impacting this transformation. As a critical step forward, we encourage broadening the current widespread screening of potentially beneficial soil microorganisms to encompass functions relevant to stimulating soil carbon stocks. Moreover, in developing these interventions, we must consider the potential ecological ramifications and uncertainties, such as incurred by the widespread introduction of homogenous inoculants and consortia, and the need for site-specificity given the extreme variation among soil habitats. Incentivization and implementation at large spatial scales could effectively harness increases in soil carbon stocks, helping to mitigate the impacts of climate change.

54 ENVIRONMENTAL SCIENCES↗

An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino-refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin-utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino-chemical industry.

09 BIOMASS FUELS↗