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Non-recombinant bacteria that produce ethanol as the primary fermentation product, associated nucleic acids and polypeptides, methods for producing ethanol using the bacteria, and kits are disclosed.
A technique for carbohydrate analysis for bioprocess samples has been developed, providing reduced analysis time compared to current practice in the biofuels R&D community. The Thermofisher CarboPac SA10 anion-exchange column enables isocratic separation of monosaccharides, sucrose and cellobiose in approximately 7 minutes. Additionally, use of a low-volume (0.2 mL) injection valve in combination with a high-volume detection cell minimizes the extent of sample dilution required to bring sugar concentrations into the linear range of the pulsed amperometric detector (PAD). Three laboratories, representing academia, industry, and government, participated in an interlaboratory study which analyzed twenty-one opportunistic samples representing biomass pretreatment, enzymatic saccharification, and fermentation samples. The technique's robustness, linearity, and interlaboratory reproducibility were evaluated and showed excellent-to-acceptable characteristics. Additionally, quantitation by the CarboPac SA10/PAD was compared with the current practice method utilizing a HPX-87P/RID. While these two methods showed good agreement a statistical comparison found significant quantitation difference between them, highlighting the difference between selective and universal detection modes.
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.
Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion and allows for the determination of optimized pretreatment conditions. Additionally, ethanol yields from a Simultaneous Saccharification and Fermentation process are improved 5-25% by treatment with a lignin-blocking polypeptide and/or protein.
The present invention provides isolated nucleic acid molecules which encode a mutant acetaldehyde-CoA/alcohol dehydrogenase or mutant alcohol dehydrogenase and confer enhanced tolerance to ethanol. The invention also provides related expression vectors, genetically engineered microorganisms having enhanced tolerance to ethanol, as well as methods of making and using such genetically modified microorganisms for production of biofuels based on fermentation of biomass materials.
Genetically engineered microorganisms have been constructed to produce succinate and malate in mineral salt media in pH-controlled batch fermentations without the addition of plasmids or foreign genes. The subject invention also provides methods of producing succinate and malate comprising the culture of genetically modified microorganisms.
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.
The invention provides recombinant bacteria, which comprise a full complement of heterologous ethanol production genes. Expression of the full complement of heterologous ethanol production genes causes the recombinant bacteria to produce ethanol as the primary fermentation product when grown in mineral salts medium, without the addition of complex nutrients. Methods for producing the recombinant bacteria and methods for producing ethanol using the recombinant bacteria are also disclosed.
Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.
The invention provides methods for decreasing lignin content and for increasing the level of fermentable carbohydrates in plants by down-regulation of the NST transcription factor. Nucleic acid constructs for down-regulation of NST are described. Transgenic plants are provided that comprise reduced lignin content. Plants described herein may be used, for example, as improved biofuel feedstock and as highly digestible forage crops. Methods for processing plant tissue and for producing ethanol by utilizing such plants are also provided.
This invention relates to the metabolic evolution of a microbial organism previously optimized for producing an organic acid in commercially significant quantities under fermentative conditions using a hexose sugar as sole source of carbon in a minimal mineral medium. As a result of this metabolic evolution, the microbial organism acquires the ability to use pentose sugars derived from cellulosic materials for its growth while retaining the original growth kinetics, the rate of organic acid production and the ability to use hexose sugars as a source of carbon. This invention also discloses the genetic change in the microorganism that confers the ability to use both the hexose and pentose sugars simultaneously in the production of commercially significant quantities of organic acids.
The JGI Fungal Genomics Program aims to scale up sequencing and analysis of fungal genomes to explore the diversity of fungi important for energy and the environment, and to promote functional studies on a system level. Combining new sequencing technologies and comparative genomics tools, JGI is now leading the world in fungal genome sequencing and analysis. Over 120 sequenced fungal genomes with analytical tools are available via MycoCosm (www.jgi.doe.gov/fungi), a web-portal for fungal biologists. Our model of interacting with user communities, unique among other sequencing centers, helps organize these communities, improves genome annotation and analysis work, and facilitates new larger-scale genomic projects. This resulted in 20 high-profile papers published in 2011 alone and contributing to the Genomics Encyclopedia of Fungi, which targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts). Our next grand challenges include larger scale exploration of fungal diversity (1000 fungal genomes), developing molecular tools for DOE-relevant model organisms, and analysis of complex systems and metagenomes.
Genomes of fungi relevant to energy and environment are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). Its key project, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts), and explores fungal diversity by means of genome sequencing and analysis. Over 150 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functional genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such parts suggested by comparative genomics and functional analysis in these areas are presented here.
Pelosinus fermentans strain R7 was isolated from Russian kaolin clays as the type strain and it can reduce Fe(III) during fermentative growth (1). Draft genome sequences for P. fermentans R7 and four strains from Hanford, Washington, USA, have been published (2–4). The P. fermentans 16S rRNA sequence dominated the lactate-based enrichment cultures from three geochemically contrasting soils from the Melton Branch Watershed, Oak Ridge, Tennessee, USA (5) and also at another stimulated, uraniumcontaminated field site near Oak Ridge (6). For the current work, strain UFO1 was isolated from pristine sediments at a background field site in Oak Ridge and characterized as facilitating U(VI) reduction and precipitation with phosphate (7).
Renewable fuel from lignocellulosic biomass has recently attracted more attention due to its environmental and the potential economic benefits over the crude oil [1]. In particular the production of fuel range hydrocarbon (HC) from alcohol generated lots of interest since the alcohol can be produced from biomass via thermochemical [2] (mixed alcohol from gasification derived synthesis gas) as well as the biochemical routes [3] (alcohol fermentation). Along with the development of ZSM5 synthesis and the discovery of methanol-to-gasoline (MTG) process by Mobil in 1970’s triggered lots of interest in research and development arena to understand the reaction mechanisms of alcohols over zeolites in particular ZSM5 [4]. More detailed research on methanol conversion was extensively reported [5] and in recent times the research work can be found on ethanol [6] and other alcohols as well but comprehensive comparison of catalyst activity and the deactivation mechanism of the conversion of various alcohols over zeolites has not been reported. The experiments were conducted on smaller alcohols such as methanol, ethanol and 1-propanol over HZSM5. The experimental results on the catalyst activity and the catalyst deactivation mechanism will be discussed.
One of the major challenges associated with algal biofuels production in a biorefinery-type setting is improving biomass utilization in its entirety, increasing the process energetic yields and providing economically viable and scalable co-product concepts. We demonstrate the effectiveness of a novel, integrated technology based on moderate temperatures and low pH to convert the carbohydrates in wet algal biomass to soluble sugars for fermentation, while making lipids more accessible for downstream extraction and leaving a protein-enriched fraction behind. This research has been highlighted in the Green Chemistry journal article mentioned above and a milestone report, and is based on the work the researchers are doing for the AOP projects Algal Biomass Conversion and Algal Biofuels Techno-economic Analysis. That work has demonstrated an advanced process for algal biofuel production that captures the value of both the algal lipids and carbohydrates for conversion to biofuels. With this process, as much as 150 GGE/ton of biomass can be produced, 2-3X more than can be produced by terrestrial feedstocks. This can also reduce the cost of biofuel production by as much as 40%. This also represents the first ever design case for the algal lipid upgrading pathway.
Executive Summary The Illinois Basin – Decatur Project (IBDP) is managed by the Midwest Geological Sequestration Consortium (MGSC) and is led by the Illinois State Geological Survey (ISGS) at the University of Illinois. The project site is located on the Archer Daniels Midland Company (ADM) property in Decatur, Illinois, and is a fully integrated carbon capture and storage (CCS) project that uses CO₂ captured from the ethanol-producing fermentation process at the ADM corn-processing plant (Finley et. al., 2013). IBDP has a goal of injecting one million tonnes of CO₂ into the basal sands of the Mt. Simon Sandstone over a three-year period. This is a multifaceted project, and this report details the planning and results of the drilling, completions, well testing, log data acquisition, and the Health, Safety, and Environment (HSE) aspects of the project. Three deep wells were planned for the IBDP: • The injection well: Injection Well #1 (CCS1); • The monitoring well (both in-zone and above seal): Verification Well #1 (VW1); and • The geophone monitoring well: Geophysical Monitoring Well #1 (GM1). The detailed plans for these wells are attached to the appendices of this document. The wells were drilled successfully with little deviation from the original plans. The biggest change from the plan to execution was the need to adjust for larger-than-expected loss of circulation in the Potosi section of the Knox Formation. The completions reports also attached to this document detail the well constructions as they were actually built. Injectivity testing was carried out, and the perforating plans were adjusted based on the results. Additional perforations and acidizing were performed as a result of the injectivity testing. The testing plans are detailed in this report along with the actual testing results. The injectivity testing results were used in the modeling and simulation efforts. Detailed HSE plans were developed and implemented during the planning and execution phases of the project. The implementation included an HSE Bridging Document, which served to unify the HSE policies of the project partners and key subcontractors. The HSE plan and actual HSE results are presented in this document. There were no recordable HSE incidents during the project. A detailed logging program was developed based on project needs. The log data were acquired in accordance with the plan, and both the plan and log results are presented in this report. Log data were heavily utilized by the research staff, modelers, reservoir engineers, and for technical and permitting efforts. 5 Several key lessons were learned during the project: • Safety in operations and execution is paramount and is only achieved through proper planning and behavior control. The certainty of this was reinforced through implementation of this lesson and the resultant flawless HSE performance during the project. • Losses of drilling fluid circulation were larger than anticipated within the Potosi Formation. Circulation was only recovered through cementing the loss zones. • When possible, minimizing complexity in permit requirements and well designs is preferable. • The size of the wells were outside of the standard experience and expertise typical within the basin, and therefore required substantial planning and ramp-up of contractors and partners to meet project objectives. • With multiple stakeholders and research partners, establishing objectives and requirements early and adhering to change request procedures throughout the project are critical to manage competing data and sampling objectives that may be detrimental to overall progress. The well construction and completion operations were successfully executed, with all wells built in a manner that achieved excellent wellbore integrity. Log planning involved a number of stakeholders and technical specialists. Data collection from logging, coring, and testing was excellent. Time and effort spent with the associated contractors and suppliers to develop a well plan beyond normal scope proved highly successful, resulting in a well-construction and completion project that surpassed expectations. The world-class HSE results also demonstrate the commitment of all stakeholders in the project. The details follow in the body of this document
Fungi play important roles across the range of current and future biofuel production processes. From crop/feedstock health to plant biomass saccharification, enzyme production to bioprocesses for producing ethanol, higher alcohols or future hydrocarbon biofuels, fungi are involved. Research and development are underway to understand the underlying biological processes and improve them to make efficient on an industrial scale. Genomics is the foundation of the systems biology approach that is being used to accelerate the research and development efforts across the spectrum of topic areas that impact biofuels production. In this review, we discuss past, current and future advances made possible by genomic analysis of the fungi that impact plant/feedstock health, degradation of lignocellulosic biomass and fermentation of sugars to ethanol, hydrocarbon biofuels and renewable chemicals.