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Bioprocess development enhanced muconate titers and productivities from mixed sugars, leading to reduced production costs and a significant decrease in GHG emissions compared to fossil carbon-based adipic acid production. Created with BioRender.com.
..beta..-Ketoadipic acid is a common intermediate in aerobic microbial aromatic catabolism that can be used as a monomer in performance-advantaged biopolymers. Here, we engineered Pseudomonas putida KT2440 to produce ..beta..-ketoadipate from glucose and xylose, the most prevalent carbohydrates derived from lignocellulosic polysaccharides. With the top-performing strain, P. putida GR038, we conducted bioprocess development using glucose and xylose as substrates in a 2 : 1 molar ratio to mimic lignocellulosic hydrolysate. Fed-batch cultivations achieved a titer of 65.8 g L-1 and a rate of 0.69 g L-1 h-1, with a C-mol yield of 0.52. Application of adsorptive in situ product recovery further improved the effective titer to 92.0 g L-1 and the rate to 0.83 g L-1 h-1, while also improving the downstream purity of ..beta..-ketoadipate from 88.3 wt% to 99.0 wt%. These results show promise towards industrial production of ..beta..-ketoadipate from lignocellulosic sugars.
Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl– and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $\$2.01$ per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($\$1.10$ to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.
Through DOE’s Bioenergy Technologies Office and the Energy Efficiency and Renewable Energy Small Business Voucher (SBV) Pilot, ZymoChem, Inc received support to work with the ABPDU to accelerate the development of a novel adipic acid (AA) bioprocess by evaluating technologies to demonstrate the following: 1) Process development of AA via ZymoChem’s carbon-conserving (C 2 ) pathway in 2L bioreactors, using industrial feedstocks, and as a salt with hexamethylenediamine (HMDA), i.e., the industrially desired form of AA as AA-HMDA is the precursor for nylon 6,6 synthesis. 2) Pilot scale aerobic fermentation at the 300 L scale (stage 1) followed by a high cell density (HCD) anaerobic resting cell biotransformation (RCB) at the 50 L scale (stage 2). 3) Demonstration of a viable recovery process at the 50 L scale for purifying the product (i.e., AA-HMDA salt) and deliver multiple kg-quantity batches of purified product for further testing.
This report summarizes the efforts undertaken by the Bioprocessing Separations Consortium since its inception in 2016. During this time, four critical challenges in biochemical and thermochemical processing were addressed.
Develop a high-yield and cost-efficient bioprocess to produce an AMP (anti-microbial peptide) in a current ABF (Agile BioFoundry) host or readily onboarded hosts. Scale up the process in stirred tank reactors and produce at least 100 grams of AMP at 1-5g/L.
Microbial biomanufacturing is important to accelerate lunar construction because it can leverage lunar material and waste streams as feedstocks to create a circular production system. In-space bio-mining and biomanufacturing using moon and asteroidal source material will enable the creation of infrastructure, produce industrial fuels and lubricants, and enable recovery of actinides and rare-earth elements (REEs) present in trace concentrations. Moreover, biomanufacturing in closed-loop systems (recycling and reuse of resources toward the establishment of a circular economy) will enable long-term lunar activities by recycling waste (CO 2 , gray water) and producing oxygen and biomaterials. Our response focuses on the use of lunar regolith and waste streams as feedstocks for protein and microbial-enabled biomining and bioprocessing to extract actinides and REEs, and to create biocomposites for lunar infrastructure. We envision an enclosed process that initiates with (1a) extracting actinides and REEs from lunar regolith using immobilized proteins, followed by (1b) creating biocomposites from the post-extracted lunar regolith for infrastructure, and (1c) cultivating diatoms and other microalgae on waste streams to harvest silica shells for incorporating into biocomposites and to generate O 2 for human respiration and/or producing refinable feedstocks. LLNL has significant expertise in all three processes and provides facilities, personnel, and expertise at the intersection of metal (lanthanide, actinide, transition) separations, purifications, biohydrometallurgy, radiobiochemistry, synthetic and systems biology, and materials science and engineering. Importantly, all three processes are relatively well-studied for Earth-based workflows and can be derisked for demonstration on the lunar surface by 2029.
Crop Protection, specifically controlling agricultural diseases and pests, is essential for crop production at every agricultural scale. Biologics hold enormous potential in this equation to decrease use of more toxic pesticides. Currently, the widespread use of biologics in crop protection is severely hampered by the lack of available tools for their mass production. To ensure biologics are regulated under EPA Biopesticide regulations, the following must be true: The biopesticide is a naturally occurring substance or structurally similar and functionally identical to a naturally occurring substance with a history of exposure to humans and the environment demonstrating minimal toxicity. Since this pertains to the marketed product it is also essential that fermentation hosts have a history of exposure and safe use by humans This research focuses on developing a novel platform for the production and delivery of the cghSAMP peptide using the filamentous fungus Aspergillus niger. A. niger is a well-established industrial microorganism, widely recognized for its strong protein secretion capabilities, high-yield production of enzymes, and classification as a generally recognized as safe (GRAS) organism. By genetically modifying A. niger to express the cghSAMP peptide transgene, we aim to leverage the fungus's robust fermentation system to produce a high volume of the antimicrobial agent. This engineered system represents a potential breakthrough in sustainable disease management, offering an environmentally friendly method for localized application or large-scale production of a biopesticide designed to mitigate the effects of citrus greening disease. The main objectives of this project were: 1. Developing a high-yield and cost-efficient bioprocess to produce an AMP in a current ABF host or readily onboarded hosts. 2. Scale up the process in stirred tank reactors and produce at least 100 grams of AMP at 1- 5g/L
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We currently understand the carbon cycle in methanogenic environments as trophic interactions revolving around interspecies H 2 transfer between organotrophs and methanogens. However, many H 2 -generating processes are thermodynamically sensitive to H 2 accumulation and can be inhibited by high H 2 concentrations produced by other metabolisms. To uncover how anaerobes combat this H 2 conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology – methanogenic bioreactors. Through analysis of 15 anaerobic digesters, we recover 1343 high-quality metagenome-assembled genomes (MAGs) and corresponding gene expression profiles for diverse uncultured lineages spanning 66 phyla and reconstruct their individual metabolic capacities. We discover that diverse uncultured organotrophic populations can drive H 2 -sensitive degradation through (i) metabolic coupling with concurrent H 2 -tolerant catabolism, (ii) unprecedented shifts from H 2 generation to CO 2 -reducing formate transfer and cytochrome/pili-mediated direct interspecies electron transfer for avoiding thermodynamic conflict, and (iii) integration of low-concentration O 2 as an ancillary thermodynamics-enhancing electron sink. Archaeal populations in situ support the above processes through novel metabolisms distinct from conventional methanogenesis – high-affinity H 2 oxidation driven by methyl-reducing methanogenesis and concomitant uptake of formate, electrons, and acetate from HS metabolism by Methanothrix. In total, synthesis of omics analyses and eco-thermodynamics reveals overlooked metabolic behavior and interactions of uncultured organisms that address a central issue in methanogenic carbon cycling, thermodynamic conflict among diverse concurrent metabolic processes.
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Potential to capture 70% of feedstock carbon while >50% of feedstock lower heating value is converted to biofuel and coproducts.
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A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.
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Some aspects of this invention provide methods and bioreactors for converting a carbon source into a lipid. In some embodiments, lipid production is carried out in an aerobic fermentor and carbon dioxide generated during lipid production is converted into a carbon substrate by CO2 fixation in an anaerobic fermentor. In some embodiments, the carbon substrate generated by CO2 fixation is used as the carbon source for lipid production, thus achieving total carbon utilization in lipid production.
Single-carbon (C1) substrates including carbon dioxide, carbon monoxide, and methane are abundantly available from natural and anthropogenic sources and present potential feedstocks for biomanufacturing. Utilizing these C1 gas feedstocks in bioprocesses for sustainable production of chemicals and fuels could prove pivotal in removing excess carbon from the atmosphere. This perspective describes the spectrum and sources of CO2, CO, and CH4 and examines emerging opportunities in microbial bioconversion and bioelectrochemical processes for these feedstocks. We discuss existing challenges in bioprocess development that currently restrict the commercialization of C1 biomanufacturing technologies. We detail different aerobic and anaerobic bioconversion approaches for C1 feedstocks employing pure and mixed cultures and examine the suitability of each scenario for producing specific molecules. Beyond strain engineering and bioprocess constraints, we address often overlooked factors that limit the development of efficient and reliable bioprocesses, including technology availability for research and safety considerations. We then discuss and recommend the necessary safety features and technological research tools for developing fast, safe, and efficient bioprocesses using gaseous feedstocks to support the scale-up and commercialization of C1 biomanufacturing technologies. This perspective provides an overview of the current scientific and industrial state of the art and offers insights into future technological needs that must be addressed to realize the potential of biomanufacturing from gaseous feedstocks. Synopsis: C1 gases offer a sustainable feedstock for biomanufacturing of fuels and chemicals. This work analyzes bioconversion methods, challenges, and safety considerations, and emphasizes the need for improved technology to enable commercialization.