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At least 1,549 records · Page 86
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Depth-discrete metagenomics reveals the roles of microbes in biogeochemical cycling in the tropical freshwater Lake Tanganyika

Lake Tanganyika (LT) is the largest tropical freshwater lake, and the largest body of anoxic freshwater on Earth’s surface. LT’s mixed oxygenated surface waters float atop a permanently anoxic layer and host rich animal biodiversity. However, little is known about microorganisms inhabiting LT’s 1470 meter deep water column and their contributions to nutrient cycling, which affect ecosystem-level function and productivity. Here, we applied genome-resolved metagenomics and environmental analyses to link specific taxa to key biogeochemical processes across a vertical depth gradient in LT. We reconstructed 523 unique metagenome-assembled genomes (MAGs) from 34 bacterial and archaeal phyla, including many rarely observed in freshwater lakes. We identified sharp contrasts in community composition and metabolic potential with an abundance of typical freshwater taxa in oxygenated mixed upper layers, and Archaea and uncultured Candidate Phyla in deep anoxic waters. Genomic capacity for nitrogen and sulfur cycling was abundant in MAGs recovered from anoxic waters, highlighting microbial contributions to the productive surface layers via recycling of upwelled nutrients, and greenhouse gases such as nitrous oxide. Overall, our study provides a blueprint for incorporation of aquatic microbial genomics in the representation of tropical freshwater lakes, especially in the context of ongoing climate change, which is predicted to bring increased stratification and anoxia to freshwater lakes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Development of the preterm infant gut microbiome: A research priority

The very low birth weight (VLBW) infant is at great risk for marked dysbiosis of the gut microbiome due to multiple factors, including physiological immaturity and prenatal/postnatal influences that disrupt the development of a normal gut flora. However, little is known about the developmental succession of the microbiota in preterm infants as they grow and mature. This review provides a synthesis of our understanding of the normal development of the infant gut microbiome and contrasts this with dysbiotic development in the VLBW infant. The role of human milk in normal gut microbial development is emphasized, along with the role of the gut microbiome in immune development and gastroenteric health. Current research provides evidence that the gut microbiome interacts extensively with many physiological systems and metabolic processes in the developing infant. However, to the best of our knowledge, there are currently no studies prospectively mapping the gut microbiome of VLBW infants through early childhood. This knowledge gap must be filled to inform a healthcare system that can provide for the growth, health, and development of VLBW infants. In conclusion, the study speculates about how the VLBW infants’ gut microbiome might function through host-microbe interactions to contribute to the sequelae of preterm birth, including its influence on growth, development, and general health of the infant host.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Beyond denitrification: The role of microbial diversity in controlling nitrous oxide reduction and soil nitrous oxide emissions

Many biotic and abiotic processes contribute to nitrous oxide (N 2 O) production in the biosphere, but N 2 O consumption in the environment has heretofore been attributed primarily to canonical denitrifying microorganisms. The nosZ genes encoding the N 2 O reductase enzyme, NosZ, responsible for N 2 O reduction to dinitrogen are now known to include two distinct groups: the well-studied Clade I which denitrifiers typically possess, and the novel Clade II possessed by diverse groups of microorganisms, most of which are non-denitrifiers. Clade II N 2 O reducers could play an important, previously unrecognized role in controlling N 2 O emissions for several reasons, including: (1) the consumption of N 2 O produced by processes other than denitrification, (2) hypothesized non-respiratory functions of NosZ as an electron sink or for N 2 O detoxification, (3) possible differing enzyme kinetics of Clade II NosZ compared to Clade I NosZ, and (4) greater nosZ gene abundance for Clade II compared to Clade I in soils of many ecosystems. Despite the potential ecological significance of Clade II NosZ, a census of 800 peer-reviewed original research articles discussing nosZ and published from 2013 to 2019 showed that the percentage of articles evaluating or mentioning Clade II nosZ increased from 5% in 2013 to only 22% in 2019. The census revealed that the slowly spreading awareness of Clade II nosZ may result in part from disciplinary silos, with the percentage of nosZ articles mentioning Clade II nosZ ranging from 0% in Agriculture and Agronomy journals to 32% in Multidisciplinary Sciences journals. In addition, inconsistent nomenclature for Clade I nosZ and Clade II nosZ, with 17 different terminologies used in the literature, may have created confusion about the two distinct groups of N 2 O reducers. Furthermore, we provide recommendations to accelerate advances in understanding the role of the diversity of N 2 O reducers in regulating soil N 2 O emissions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

iVirus 2.0: Cyberinfrastructure-supported tools and data to power DNA virus ecology

Microbes drive myriad ecosystem processes, but under strong influence from viruses. Because studying viruses in complex systems requires different tools than those for microbes, they remain underexplored. To combat this, we previously aggregated double-stranded DNA (dsDNA) virus analysis capabilities and resources into ‘iVirus’ on the CyVerse collaborative cyberinfrastructure. Here we substantially expand iVirus’s functionality and accessibility, to iVirus 2.0, as follows. First, core iVirus apps were integrated into the Department of Energy’s Systems Biology KnowledgeBase (KBase) to provide an additional analytical platform. Second, at CyVerse, 20 software tools (apps) were upgraded or added as new tools and capabilities. Third, nearly 20-fold more sequence reads were aggregated to capture new data and environments. Finally, documentation, as “live” protocols, was updated to maximize user interaction with and contribution to infrastructure development. Together, iVirus 2.0 serves as a uniquely central and accessible analytical platform for studying how viruses, particularly dsDNA viruses, impact diverse microbial ecosystems.

Bolduc, Benjamin (ORCID:0000000324200755)↗
DOE OSTI2017

Enhanced Production of Bioethanol by Fermentation of Autohydrolyzed and C4mimOAc-Treated Sugarcane Bagasse Employing Various Yeast Strains

Natural dissolved organic matter (DOM) affects mercury (Hg) redox reactions and anaerobic microbial Hg methylation in the environment. Several studies have shown that DOM can enhance Hg methylation, especially under sulfidic conditions, whereas others show that DOM inhibits Hg methylation due to strong Hg-DOM complexation. Here, we investigated and compared the effects of DOM on Hg methylation by an iron-reducing bacterium Geobacter sulfurreducens PCA and a sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 under non-sulfidic conditions. The methylation experiment was performed with washed cells either in the absence or presence of DOM or glutathione, both of which form strong complexes with Hg via thiol-functional groups. DOM was found to greatly inhibit Hg methylation by G. Sulfurreducens PCA but enhance Hg methylation by D. desulfuricans ND132 cells with increasing DOM concentration. These strain-dependent opposing effects of DOM were also observed with glutathione, suggesting that thiols in DOM likely played an essential role in affecting cell Hg uptake and methylation. Additionally, DOM and glutathione decreased Hg sorption by G. sulfurreducens PCA, but not by D. desulfuricans ND132 cells, demonstrating that ND132 has a higher affinity to sorb or take up Hg than the PCA strain. Our observations indicate that DOM effects on Hg methylation are bacterial strain specific, depend on the DOM:Hg ratio or site-specific conditions, and may thus offer new insights into the role of DOM in methylmercury production in the environment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Phage-specific metabolic reprogramming of virocells

Ocean viruses are abundant and infect 20–40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage–host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage–host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.

Environmental Sciences & Ecology↗
DOE OSTI2022

The IMG/M data management and analysis system v.7: content updates and new features

The Integrated Microbial Genomes & Microbiomes system at the Department of Energy (DOE) Joint Genome Institute (JGI) continues to provide support for users to perform comparative analysis of isolate and single cell genomes, metagenomes, and metatranscriptomes. In addition to datasets produced by the JGI, IMG v.7 also includes datasets imported from public sources such as NCBI Genbank, SRA, and the DOE National Microbiome Data Collaborative (NMDC), or submitted by external users. In the past couple years, we have continued our effort to help the user community by improving the annotation pipeline, upgrading the contents with new reference database versions, and adding new analysis functionalities such as advanced scaffold search, Average Nucleotide Identity (ANI) for high-quality metagenome bins, new cassette search, improved gene neighborhood display, and improvements to metatranscriptome data display and analysis. Here, we also extended the collaboration and integration efforts with other DOE-funded projects such as NMDC and DOE Biology Knowledgebase (KBase).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

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↗
DOE OSTI2020

Microbial Diversity Associated with the Pollen Stores of Captive-Bred Bumble Bee Colonies

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms of infection. The shared core microbiota across both commercial supplier sources consisted of two ubiquitous bee-associated groups, Lactobacillus and Wickerhamiella/Starmerella clade yeasts that potentially contribute to the beneficial function of the microbiome of bumble bee pollen provisions.

16S rRNA gene↗
DOE OSTI2019

Metagenomic Evaluation of Bacterial and Fungal Assemblages Enriched within Diffusion Chambers and Microbial Traps Containing Uraniferous Soils

Despite significant technological advancements in the field of microbial ecology, cultivation and subsequent isolation of the vast majority of environmental microorganisms continues to pose challenges. Isolation of the environmental microbiomes is prerequisite to better understand a myriad of ecosystem services they provide, such as bioremediation of contaminants. Towards this end, in this culturomics study, we evaluated the colonization of soil bacterial and fungal communities within diffusion chambers (DC) and microbial traps (MT) established using uraniferous soils collected from a historically contaminated soil from Aiken, USA. Microbial assemblages were compared between the DC and MT relative to the native soils using amplicon based metagenomic and bioinformatic analysis. The overall rationale of this study is that DC and MT growth chambers provide the optimum conditions under which desired microbiota, identified in a previous study to serve as the “core” microbiomes, will proliferate, leading to their successful isolation. Specifically, the core microbiomes consisted of assemblages of bacteria (Burkholderia spp.) and fungi (Penicillium spp.), respectively. The findings from this study further supported previous data such that the abundance and diversity of the desired “core” microbiomes significantly increased as a function of enrichments over three consecutive generations of DC and MT, respectively. Metagenomic analysis of the DC/MT generations also revealed that enrichment and stable populations of the desired “core” bacterial and fungal microbiomes develop within the first 20 days of incubation and the practice of subsequent transfers for second and third generations, as is standard in previous studies, may be unnecessary. As a cost and time cutting measure, this study recommends running the DC/MT chambers for only a 20-day time period, as opposed to previous studies, which were run for months. In summation, it was concluded that, using the diffusion chamber-based enrichment techniques, growth of desired microbiota possessing environmentally relevant functions can be achieved in a much shorter time frame than has been previously shown.

Jaswal, Rajneesh (ORCID:0000000217841805)↗
DOE OSTI2016

‘ Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. Here, we describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L -1 d -1 . The DCM degrader attained 5.25 × 10 7 ± 1.0 × 10 7 cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Furthermore, based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Methanobactin from methanotrophs: genetics, structure, function and potential applications

ABSTRACT Aerobic methane-oxidizing bacteria of the Alphaproteobacteria have been found to express a novel ribosomally synthesized post-translationally modified polypeptide (RiPP) termed methanobactin (MB). The primary function of MB in these microbes appears to be for copper uptake, but MB has been shown to have multiple capabilities, including oxidase, superoxide dismutase and hydrogen peroxide reductase activities, the ability to detoxify mercury species, as well as acting as an antimicrobial agent. Herein, we describe the diversity of known MBs as well as the genetics underlying MB biosynthesis. We further propose based on bioinformatics analyses that some methanotrophs may produce novel forms of MB that have yet to be characterized. We also discuss recent findings documenting that MBs play an important role in controlling copper availability to the broader microbial community, and as a result can strongly affect the activity of microbes that require copper for important enzymatic transformations, e.g. conversion of nitrous oxide to dinitrogen. Finally, we describe procedures for the detection/purification of MB, as well as potential medical and industrial applications of this intriguing RiPP.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2016

Biogeographic patterns of soil diazotrophic communities across six forests in the North America

Soil diazotrophs play important roles in ecosystem functioning by converting atmospheric N 2 into biologically available ammonium. However, the diversity and distribution of soil diazotrophic communities in different forests and whether they follow biogeographic patterns similar to macroorganisms still remain unclear. By sequencing nifH gene amplicons, we surveyed the diversity, structure and biogeographic patterns of soil diazotrophic communities across six North American forests (126 nested samples). Our results showed that each forest harboured markedly different soil diazotrophic communities and that these communities followed traditional biogeographic patterns similar to plant and animal communities, including the taxa–area relationship (TAR) and latitudinal diversity gradient. Significantly higher community diversity and lower microbial spatial turnover rates (i.e. z-values) were found for rainforests (~0.06) than temperate forests (~0.1). The gradient pattern of TARs and community diversity was strongly correlated (r 2 > 0.5) with latitude, annual mean temperature, plant species richness and precipitation, and weakly correlated (r 2 < 0.25) with pH and soil moisture. Here, this study suggests that even microbial subcommunities (e.g. soil diazotrophs) follow general biogeographic patterns (e.g. TAR, latitudinal diversity gradient), and indicates that the metabolic theory of ecology and habitat heterogeneity may be the major underlying ecological mechanisms shaping the biogeographic patterns of soil diazotrophic communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Inferring the Contribution of Microbial Taxa and Organic Matter Molecular Formulas to Ecological Assembly

Understanding the mechanisms underlying the assembly of communities has long been the goal of many ecological studies. While several studies have evaluated community wide ecological assembly, fewer have focused on investigating the impacts of individual members within a community or assemblage on ecological assembly. Here, we adapted a previous null model β-nearest taxon index (βNTI) to measure the contribution of individual features within an ecological community to overall assembly. This new metric, called feature-level βNTI (βNTI feat ), enables researchers to determine whether ecological features (e.g., individual microbial taxa) contribute to divergence, convergence, or have insignificant impacts across spatiotemporally resolved metacommunities or meta-assemblages. Using βNTI feat , we revealed that unclassified microbial lineages often contributed to community divergence while diverse groups (e.g., Crenarchaeota, Alphaproteobacteria, and Gammaproteobacteria) contributed to convergence. We also demonstrate that βNTI feat can be extended to other ecological assemblages such as organic molecules comprising organic matter (OM) pools. OM had more inconsistent trends compared to the microbial community though CHO-containing molecular formulas often contributed to convergence, while nitrogen and phosphorus-containing formulas contributed to both convergence and divergence. A network analysis was used to relate βNTI feat values from the putatively active microbial community and the OM assemblage and examine potentially common contributions to ecological assembly across different communities/assemblages. This analysis revealed that P-containing formulas often contributed to convergence/divergence separately from other ecological features and N-containing formulas often contributed to assembly in coordination with microorganisms. Additionally, members of Family Geobacteraceae were often observed to contribute to convergence/divergence in conjunction with both N- and P-containing formulas, suggesting a coordinated ecological role for family members and the nitrogen/phosphorus cycle. Overall, we show that βNTI feat offers opportunities to investigate the community or assemblage members, which shape the phylogenetic or functional landscape, and demonstrate the potential to evaluate potential points of coordination across various community types.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Structural Basis for Metallic-Like Conductivity in Microbial Nanowires

Direct measurement of multiple physical properties of Geobacter sulfurreducens pili have demonstrated that they possess metallic-like conductivity, but several studies have suggested that metallic-like conductivity is unlikely based on the structures of the G. sulfurreducens pilus predicted from homology models. In order to further evaluate this discrepancy, pili were examined with synchrotron X-ray microdiffraction and rocking-curve X-ray diffraction. Both techniques revealed a periodic 3.2-Å spacing in conductive, wild-type G. sulfurreducens pili that was missing in the nonconductive pili of strain Aro5, which lack key aromatic acids required for conductivity. The intensity of the 3.2-Å peak increased 100-fold when the pH was shifted from 10.5 to 2, corresponding with a previously reported 100-fold increase in pilus conductivity with this pH change. These results suggest a clear structure-function correlation for metallic-like conductivity that can be attributed to overlapping π-orbitals of aromatic amino acids. A homology model of the G. sulfurreducens pilus was constructed with a Pseudomonas aeruginosa pilus model as a template as an alternative to previous models, which were based on a Neisseria gonorrhoeae pilus structure. This alternative model predicted that aromatic amino acids in G. sulfurreducens pili are packed within 3 to 4 Å, consistent with the experimental results. Thus, the predictions of homology modeling are highly sensitive to assumptions inherent in the model construction. Finally, the experimental results reported here further support the concept that the pili of G. sulfurreducens represent a novel class of electronically functional proteins in which aromatic amino acids promote long-distance electron transport.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Impact of Polymicrobial Infection on Fitness of Streptococcus gordonii In Vivo

Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis. In the context of a monoinfection, conditionally essential genes were widely distributed among functional pathways. Coinfection with P. gingivalis almost completely changed the nature of in vivo gene essentiality. Community-dependent essential (CoDE) genes under the coinfection condition were primarily related to DNA replication, transcription, and translation, indicating that robust growth and replication are required to survive with P. gingivalis in vivo. Interestingly, a group of genes in an operon encoding streptococcal receptor polysaccharide (RPS) were associated with decreased fitness of S. gordonii in a coinfection with P. gingivalis. Individual deletion of two of these genes (SGO_2020 and SGO_2024) resulted in the loss of RPS production by S. gordonii and increased susceptibility to killing by neutrophils. P. gingivalis protected the RPS mutants by inhibiting neutrophil recruitment, degranulation, and neutrophil extracellular trap (NET) formation. These results provide insight into genes and functions that are important for S. gordonii survival in vivo and the nature of polymicrobial synergy with P. gingivalis. Furthermore, we show that RPS-mediated immune protection in S. gordonii is dispensable and detrimental in the presence of a synergistic partner species that can interfere with neutrophil killing mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Final Technical Report: Role of Methanotrophs in Metal Mobilization, Metal Immobilization and Mineral Weathering: Effects on the In Situ Microbial Community and the Sustainability of Subsurface Water Systems

Activities from this DOE sponsored project can be divided into four broad areas: (1) investigations into the potential of methanobactin, a biogenic metal-binding agent produced by methanotrophs, in mitigating mercury toxicity; (2) elucidation of the genetic basis for methanobactin synthesis from methanotrophs; (3) examination of differential gene expression of M. trichosporium OB3b when grown in the presence of varying amounts of copper and/or cerium, and (4) collection and characterization of soil cores from Savannah River Test Site to determine the ubiquity of methanobactin producing methanotrophs. From these efforts, we have conclusively shown that methanobactin can strongly bind mercury as Hg[II], and in so doing significantly reduce the toxicity of this metal to microbes. Further, we have deduced the genetic basis of methanobactin production in methanotrophs, enabling us to construct mutants such that we can now ascribe function to different genes as well as propose a pathway for methanobactin biosynthesis. We have also clear evidence that copper and cerium (as an example of a rare earth element) dramatically affect gene expression in methanotrophs, and thus have an important impact on the activity and application of these microbes to a variety of environmental and industrial issues. Finally, we successfully isolated one methanotroph from the deep subsurface of the Savannah River Test Site and characterized the ability of different forms of methanobactin to mobilize copper and mercury from these soils.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2016

Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate (C 2 C 1 Im][OAc] ) for pretreatment of switchgrass biomass. [C 2 C 1 Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C 2 C 1 Im]-tolerant E. coli strain, where [C 2 C 1 Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C 2 C 1 Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C 2 C 1 Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C 2 C 1 Im]-tolerant E. coli strain so as to secrete this [C 2 C 1 Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.

09 BIOMASS FUELS↗