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Simulation results for "Integration of Plant and Microbial Oil Processing at Oilcane Biorefineries for More Sustainable Biofuel Production" publication.
Microbial oils are a sustainable biomass-derived substitute for liquid fuels and vegetable oils. Oilcane, an engineered sugarcane with superior feedstock characteristics for biodiesel production, is a promising candidate for bioconversion. This study describes the processing of oilcane stems into juice and hydrothermally pretreated lignocellulosic hydrolysate and their valorization to ethanol and microbial oil using Saccharomyces cerevisiae and engineered Rhodosporidium toruloides strains, respectively. A bioethanol titer of 106 g/L was obtained from S. cerevisiae grown on oilcane juice in a 3 L fermenter, and a lipid titer of 8.8 g/L was obtained from R. toruloides grown on oilcane hydrolysate in a 75 L fermenter. Oil was extracted from the R. toruloides cells using supercritical CO2, and the observed fatty acid profile was consistent with previous studies on this strain. These results demonstrate the feasibility of pilot-scale lipid production from oilcane hydrolysate as part of an integrated bioconversion strategy.
Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca 2+ -dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this β-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other β-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.
The goal of this project was to develop a synthetic microbial consortium consisting of a lactic acid bacterium, a carboxydotrophic acetogen, and a solventogenic clostridia for the production of n butanol, an advanced biofuel and industrial chemical, from lignocellulose sugars (mainly glucose and xylose) and formate (produced from CO 2 by electrochemical reduction) in an integrated bioprocess (biorefinery), which can provide an effective solution to the technical challenges in developing energy and carbon optimized synthesis for the bioeconomy and achieve the program objectives of ARPA-E. The project focused on the design, modeling and construction of synthetic microbial consortia consisting of three bacterial species to maximize carbon conversion and butanol production with a 100% theoretical product yield from glucose and zero or negative CO 2 emission.
Switchgrass (Panicum virgatum) is a model perennial grass for bioenergy production that can be productive in agricultural lands that are not suitable for food production. There is growing interest in whether its associated microbiome may be adaptive in low- or no-input cultivation systems. However, the relative impact of plant genotype and soil factors on plant microbiome and biomass are a challenge to decouple. To address this, a common garden greenhouse experiment was carried out using six common switchgrass genotypes, which were each grown in four different marginal soils collected from long-term bioenergy research sites in Michigan and Wisconsin. We characterized the fungal and bacterial root communities with high-throughput amplicon sequencing of the ITS and 16S rDNA markers, and collected phenological plant traits during plant growth, as well as soil chemical traits. At harvest, we measured the total plant aerial dry biomass. Significant differences in richness and Shannon diversity across soils but not between plant genotypes were found. Generalized linear models showed an interaction between soil and genotype for fungal richness but not for bacterial richness. Community structure was also strongly shaped by soil origin and soil origin × plant genotype interactions. Overall, plant genotype effects were significant but low. Random Forest models indicate that important factors impacting switchgrass biomass included NO 3 – , Ca 2+ , PO 4 3– , and microbial biodiversity. We identified 54 fungal and 52 bacterial predictors of plant aerial biomass, which included several operational taxonomic units belonging to Glomeraceae and Rhizobiaceae, fungal and bacterial lineages that are involved in provisioning nutrients to plants.
Abstract Desert tortoises play ecologically significant roles, including plant seed dispersal and mineral cycling, and yet little is known about microbial members that are critical to their gut and overall health. Tortoises consume recalcitrant plant material, which their gut microbiota degrades and converts into usable metabolites and nutrients for the tortoise. Findings from tortoise gut microbiomes may translate well into biotechnological applications as these microbes have evolved to efficiently degrade recalcitrant substrates and generate useful products. In this study, we cultivated microbial communities from desert tortoise fecal samples following a targeted anaerobic enrichment for microbes involved in deconstruction and utilization of plant biomass. We employed 16S rRNA amplicon sequencing to compare cultivated communities to initial fecal source material and found high abundances of Firmicutes and Bacteroidota typically associated with biomass deconstruction in all cultivated samples. Significantly decreased microbial diversity was observed in the cultivated microbial communities, yet several key taxa thrived in lignocellulose enrichments, includingLachnospiraceaeandEnterococcus. Additionally, cultivated communities produced short-chain fatty acids under anaerobic conditions, and their growth and metabolic output provide evidence of their viability in the initial fecal communities. Overall, this study adds to the limited understanding of reptilian herbivore microbiota, and offers a path towards biotechnological translation based on the ability of the cultivated communities to convert lignocellulose directly to acetate, propionate, and butyrate.
Development of microorganisms into mature bioproduction host strains has typically been a slow and circuitous process, wherein multiple groups apply disparate approaches with minimal coordination over decades. To help organize and streamline host development efforts, we introduce the Tier System for Host Development, a conceptual model and guide for developing microbial hosts that can ultimately lead to a systematic, standardized, less expensive, and more rapid workflow. The Tier System is made up of three Tiers, each consisting of a unique set of strain development Targets, including experimental tools, strain properties, experimental information, and process models. By introducing the Tier System, we hope to improve host development activities through standardization and systematization pertaining to nontraditional chassis organisms.
Indigoidine is a bioadvantaged platform molecule with diverse applications, including use as a textile dye, biotransistor, biosolar cell, biosensor, and food coloring. There are multiple microbial hosts and carbon sources that can be used and optimized for its production, yet there is limited guidance for which options have the greatest commercial potential. Here, we consider five different host microbes and combine genome-scale metabolic models with techno-economic and lifecycle assessment models. Pseudomonas putida currently outperforms synthetic indigo production and other indigoidine-producing hosts, using glucose, xylose, and lignin-derived aromatics to produce indigoidine at a minimum selling price of $2.9/kg and a greenhouse gas (GHG) footprint of 3.5 kgCO 2e /kg. Optimizing pathways-achieving 90% of the theoretical indigoidine yield from sugars and aromatics-can reduce costs 6-7-fold and GHG emissions 3-10-fold. From a cost perspective, microbes that co-utilize aromatics are advantageous, while selecting hosts that coproduce other value-added molecules can reduce GHG emissions. System-wide improvements and the use of a low-cost, low-carbon nitrogen source are crucial for commercial viability in all cases.
The growing concerns about climate change and energy security are driving the development of bio-based technologies to produce renewable liquid fuels and chemicals. Ionic liquids (ILs) have demonstrated to be promising solvents to pretreat lignocellulosic residues, promoting efficient enzymatic hydrolysis of lignocellulosic carbohydrates into sugars, which can be further used by microorganisms to produce biofuels and other value-added chemicals. Despite their unique properties to effectively deconstruct plant cell walls, ILs show strong interactions with the pretreated biomass, and their presence is often inhibitory to cellulolytic enzymes and microorganisms. The most advanced biorefinery concepts based on IL pretreatments focus on the development of more biocompatible ILs and more robust microbial strains with higher tolerance to ILs. This chapter provides an overview and a discussion over the main efforts performed on the screening and development of IL-tolerant microbial strains, as well as in more biocompatible IL pretreatment methods. These early research advancements in this field offer a baseline and a platform for future research with the goal of improving the sustainability and economic viability of IL pretreatment-based biorefineries.
Abstract Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass‐sensing RsgI‐type anti‐σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti‐σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn‐Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti‐σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.
In this work, we describe genetic tools and techniques for engineering Trichoderma reesei for the production of farnesene. Enhanced production of farnesene was used as an example of this methodology; as were the overexpression of a key enzyme, HMGS, in the MVA pathway.
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.
The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.
The concept of mass-specific power (MSP) quantitatively relates energy flux to the biomass that flux can sustain. We sought to assess the range and distribution of MSP as expressed at both the organism and biosphere level. The former serves to establish what is possible, and what predominates, in physiological terms; the latter reflects the expression of that physiological potential in an environmental context. To assess the potential range of MSP across a diversity of organisms and physiological states, we compiled a data set of more than 10,000 individual MSP measurements, encompassing more than 2900 unique species and spanning 22 orders of magnitude in body mass. Across the data set, MSP varies over six orders of magnitude, from 3 x 10-5 to 49 Watts per gram biomass carbon (W/gC), but the majority of measured values fall within a relatively narrow range of 0.018 ± 6.4-fold W/gC. It is well documented, and clearly expressed in the data set, that MSP exhibits a strong mass dependence within specific taxa (e.g., birds, mammals, and many others); however, MSP exhibits no mass dependence when considering the data set overall. To assess the expression of MSP in environmental context, we estimated energy utilization rates within eight distinct components of the global biosphere, and for the biosphere overall, and used recently published estimates of biomass within those components to compute MSP. We estimate MSP of 0.005 W/gC for the global biosphere as a whole. The close agreement with the organism-level all-species mean (within 4-fold) is noteworthy because the organism-level data set is dominated in statistical terms by animals, while the global biosphere is dominated in mass terms by trees and microorganisms. The various components of the microbe-dominated marine biosphere exhibit a systematic 5 order of magnitude decrease in MSP from the top of the water column (marine primary producers: 0.9 W/gC) to the depths of the sediment column (> 1m sediment biota: 3 x 10-6 W/gC). Biomass turnover rates, combined with the microbial capacity for both high specific growth rates and low maintenance rates, appear to underlie this large range in MSP, which is not expressed to the same extent in the terrestrial biosphere.
Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method, named ModCell-HPC, that can design modular cells for large libraries with hundreds of products with a highly-parallel and multi-objective evolutionary algorithm and enable us to elucidate modular design properties. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner with different types of fermentable sugars. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells, involving not only in the removal of major by-products but also modification of branch points in the central metabolism. We further found that the effect of various sugar degradation on redox metabolism results in lower compatibility between a modular cell and production modules for growth on pentoses than hexoses. To better characterize the degree of compatibility, we developed a method to calculate the minimal set cover, identifying that only three modular cells are all needed to couple with up 85 compatible production modules. By determining the unknown compatibility contribution metric, we further elucidated the design features that allow an existing modular cell to be re-purposed towards production of new molecules. Altogether, ModCell-HPC is a useful tool for understanding modularity of biological systems and guiding more efficient and generalizable design of modular cells that help reduce research and development cost in biocatalysis.
Crewed missions to Mars and beyond pose new challenges for the crew and the technological solutions they depend upon. Crucial considerations in designing the next generation of Environmental Control and Life Support Systems (ECLSS) include enhanced reliability and autonomy, given the challenges of resupply and resource recovery in remote missions. One significant challenge faced in various sectors of the International Space Station and ECLSS is the phenomena of microbial growth, particularly biofilm formation. As such, microbial growth is a critical consideration in the design phase of new ECLSS systems. Two novel CO2 removal systems—Liquid-Amines CO2 Removal (LACR) and Cold-Surface CO2 Deposition (CDep)—are currently under development at the NASA Ames Research Center to meet the needs of extended space travel. In this presentation, the authors aim to make a risk assessment of microbial growth in LACR and CDep through: 1) a comprehensive literature review to identify LACR and CDep components most vulnerable to microbial contamination and 2) wet lab testing commencing with a subscale test of the most susceptible component of CDep. The literature review revealed that the hollow-fiber Liqui-Cel membrane employed in CDep exhibits the highest susceptibility to microbial growth. Our wet lab tests corroborated these findings, as airborne microbes introduced into the membrane led to considerable biomass accumulation. These results indicate a significant risk of microbial invasion into the membranes. To prevent microbial growth in CDep or other CO2 removal systems, proper ECLSS arrangement is essential—a CO2 removal system should remain behind the charcoal-HEPA filters, condensing heat exchanger, and Trace Contaminant Control System. In summary, anticipating potential microbial interactions within ECLSS for remote crewed missions is vital to mission success. It should, therefore, be included as one of the many factors when designing future ECLSS systems to perform reliably and consistently to support astronaut life.
Microbial inoculants can increase the yield of cultivated crops and are successful in independent trials; however, efficacy drops in large-scale applications due to insufficient consideration of microbial community dynamics. The structure of microbiomes, in addition to the impact of individual taxa, is an important factor to consider when designing growth-promoting inoculants. Here, we investigate the microbial network and community assembly patterns of Macrocystis pyrifera gametophyte germplasm cultures (collectively referred to as a “seedbank”) used to cultivate an offshore farm in Santa Barbara, California, and identify network features associated with increased biomass of mature sporophytes. We found that [1] several network features, such as clustering coefficient and edge ratios, significantly vary with biomass outcomes; [2] gametophytes that become low- or high-biomass sporophytes have different hub taxa; and [3] microbial community assembly of gametophyte germplasm cultures is niche-driven. Overall, this study describes microbial community dynamics in M . pyrifera germplasm cultures and ultimately supports the development of early life stage inoculants that can be used on seaweed cultivars to increase biomass yield.
In the last few decades, lignocellulosic biomass has attracted substantial interest as a feedstock for fermentative production of fuels and other commodity chemicals due to its wide availability and low cost. However, lignocellulose has innate complexity and recalcitrance to biodegradation. In natural environments, effective plant biomass decay is obtained by synergistic activity of complex microbial communities.