DOE JBEI team boosts methyl ketone production from E. coli 160-fold; advanced biofuel or blendstock

2 December 2014

In 2012, researchers at the US Department of Energy’s Joint BioEnergy Institute (JBEI) engineered Escherichia coli (E. coli) bacteria to overproduce from glucose saturated and monounsaturated aliphatic methyl ketones in the C₁₁ to C₁₅ (diesel) range from glucose. In subsequent tests, these methyl ketones yielded high cetane numbers, making them promising candidates for the production of advanced biofuels or blendstocks. (Earlier post.)

Now, after further genetic modifications of the bacteria, they have managed to boost the E.coli’s methyl ketone production 160-fold. A paper describing this work is published in the journal Metabolic Engineering.

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We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene.

Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.

While the team is encouraged about such a large improvement in methyl ketone production with a relatively small number of genetic modifications, said Harry Beller, a JBEI microbiologist who led this and the earlier study, they believe they can further improve production using the knowledge gained from in vitro studies of the novel metabolic pathway.

Methyl ketones are naturally occurring compounds discovered more than a century ago in the aromatic evergreen plant known as rue. Since then they’ve been found to be common in tomatoes and other plants, as well as insects and microorganisms. Today they are used to provide scents in essential oils and flavoring in cheese and other dairy products. Although native E. coli make virtually undetectable quantities of methyl ketones, Beller, co-author Ee-Been Goh and their colleagues have been able to overcome this deficiency using the tools of synthetic biology.

Although the improved production is still not at a commercial level in the biofuel market, it is near a commercial level for use in flavor and fragrances, where certain methyl ketones are much more highly valued than they would be in the biofuel market. It may be possible for a company to sell a small percentage of methyl ketones in the flavor and fragrance market and use the profits to enhance the economic viability of the production of methyl ketones as biofuels.

The in vitro studies carried out by Beller and Goh provided insights into the pathway, some of which point to even further production gains. One key finding was the confirmation that a decarboxylase enzyme is not required for this methyl ketone pathway.

Several different metabolic pathways have been developed in the past couple of years for methyl ketone production in E. coli, a couple of which use decarboxylase enzymes to catalyze the last step of the pathway. Our methyl ketone pathway is performing quite a bit better than these other pathways, but it does not include a native or added decarboxylase.

The in vitro studies also addressed concerns about the FadM enzyme being somewhat “promiscuous” in its hydrolyzing (thioesterase) activities. Beller and Goh found that FadM can act on intermediates in the methyl ketone pathway and effectively reduce the flux of carbon to the final methyl ketone products. However, they say that with some informed metabolic engineering, this need not be a problem and knowledge of the phenomenon could even be used to enhance production.

In all likelihood, there is a sweet spot in the level of expression of the FadM enzyme that will allow for maximal production of methyl ketones without siphoning away metabolic intermediates.

This research was supported by JBEI through the US Department of Energy’s Office of Science.

Resources

• Ee-Been Goh, Edward E.K. Baidoo, Helcio Burd, Taek Soon Lee, Jay D. Keasling, Harry R. Beller (2014) “Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies,” Metabolic Engineering, Volume 26, Pages 67-76 doi: 10.1016/j.ymben.2014.09.003

EDGE3 Technologies Honda RD Americas awarded patent for HMI system combining gesture and voice recognition

2 December 2014

EDGE3 Technologies, an advanced vision analytics company, and Honda RD Americas have been awarded US patent number 8,744,645 for their jointly developed in-vehicle human machine interface (HMI) system which combines voice and gesture recognition. The core technology, an integrated vision analytics system developed by EDGE3, is a result of years of collaboration between EDGE3 and Honda RD which up until now has been kept confidential by both organizations.

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The HMI system will allow drivers more accurately and naturally to control their automobile’s infotainment and climate systems. By providing a secondary command input, the system differentiates between intended and unintentional commands while eliminating many of the problems which have plagued traditional voice command systems, such as false positives.

The project incorporates one of the most advanced computational stereo cameras for 3D machine vision together with an advanced artificial intelligence platform into a fully integrated and embeddable system which runs on a standard system on a chip (SoC).

From the onset, one of the goals of the collaboration was to leverage existing off the shelf sensors, chips and hardware to construct a world class, automotive grade HMI system.

This system is a definite game-changer for the global automotive and machine vision industry because it fundamentally modernizes how automobiles can respond to human behavior. Until now, machine vision was limited to use cases where the environment was extremely controlled and the computational requirements were unlimited. With this patented technology and other technology, we can bring sophisticated vision analytics to any platform, regardless of lighting conditions and environmental variability. We are honored to work on this project with Honda RD, which for years has been a visionary organization with respect to the introduction of new automotive technologies.

Novel single-site gold WGS catalysts may offer pathway to lower-cost production of hydrogen, fuels and chemicals

2 December 2014

A team of researchers from universities and national laboratories led by Tufts University has developed catalysts composed of a unique structure of single gold atoms bound by oxygen to several sodium or potassium atoms and supported on non-reactive silica materials. This single-site gold species is active for the low-temperature (

They thus have found that gold is similar to platinum in creating –O and –OH linkages with more than eight alkali ions and establishing an active site on various supports. This finding paves the way for using earth-abundant supports to disperse and to stabilize precious metal atoms with alkali additives for the WGS and potentially other fuel processing reactions. The result could be lower costs. A paper describing their work is now published in Science Express.

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The water-gas shift (WGS) reaction (CO + H₂O = CO₂+ H₂) is an important reaction for hydrogen upgrading during fuel gas processing. Emerging applications in fuel cells require active, non-pyrophoric, and cost-effective catalysts. Along with a new group of platinum catalysts with atomically dispersed Pt sites to maximize activity and catalytic efficiency, the lower apparent activation energy E_a for the WGS reaction (~45 kJ/mol) for gold (Au) vs. ~75 kJ/mol for platinum, can be exploited for low-temperature WGS and other reactions. Low-temperature activity is important to avoid multiple-treatment units in practical low-temperature PEM fuel cell systems, whereby the deleterious CO should be totally removed for stable, long-term operation.

… We now show how to use alkali addition to activate and stabilize atomic Au for the WGS reaction even on inert zeolite (KLTL) and mesoporous [Si]MCM-41 silica materials and measure activity comparable to that of Au on reducible oxide supports, and with good stability up to 200 °C.

In 2010, the Tufts group reported in another paper in Science that alkali ions (sodium or potassium) added in small amounts activate platinum adsorbed on alumina or silica for the low-temperature water-gas shift (WGS) reaction. The alkali ion–associated surface OH groups were activated by CO at low temperatures (~100 °C) in the presence of atomically dispersed platinum. These findings were useful for the design of highly active and stable WGS catalysts containing only trace amounts of a precious metal without the need for a reducible oxide support such as ceria.

Senior author Maria Flytzani-Stephanopoulos, the Robert and Marcy Haber Endowed Professor in Energy Sustainability and professor in the Department of Chemical and Biological Engineering at Tufts, said that the new research suggests single precious metal atoms stabilized with alkali ions may be the only important catalyst sites for other catalytic reactions.

The just-published research describes how single gold atoms dispersed on non-reactive supports based on silica materials can be stabilized with alkali ions. As long as the gold atoms, or cations, are stabilized in a single-site form configuration, irrespective of the type of support, the precious metal will be stable and operate for many hours at a range of practical temperatures.

This novel atomic-scale catalyst configuration achieves the maximum efficiency and utilization of the gold. Our work showed that these single-site gold cations were active for the low-temperature water-gas shift reaction and stable in operation at temperatures as high as 200 °C. Armed with this new understanding, practitioners will be able to design catalysts using just the necessary amount of the precious metals like gold and platinum, dramatically cutting down the catalyst cost in fuels and chemicals production processes.

Paper co-authors Professor Manos Mavrikakis at the University of Wisconsin-Madison and Assistant Professor Ye Xu at Louisiana State University used theoretical calculations to predict the stability and thermochemical properties of the single-site configuration.

Researchers Larry Allard at Oak Ridge National Laboratory and Sungsik Lee at Argonne National Laboratory used atomic resolution electron microscopy and x-ray absorption spectroscopies, respectively, to demonstrate the existence and stability of the single-site gold species. Co-author Jun Huang, a lecturer at the University of Sydney, synthesized and characterized the silica materials used as supports. Several graduate students were involved in all aspects of the research both at Tufts and the University of Wisconsin-Madison.

This research is primarily supported by the US Department of Energy under grant #DE-FG02-05ER15730.

Resources

• Ming Yang, Sha Li, Yuan Wang, Jeffrey A. Herron, Ye Xu, Lawrence F. Allard, Sungsik Lee, Jun Huang, Manos Mavrikakis, and Maria Flytzani-Stephanopoulos (2014) “Catalytically active Au-O(OH)_x— species stabilized by alkali ions on zeolites and mesoporous oxides” Science doi: 10.1126/science.1260526

• Yanping Zhai, Danny Pierre, Rui Si, Weiling Deng, Peter Ferrin, Anand U. Nilekar, Guowen Peng, Jeffrey A. Herron, David C. Bell, Howard Saltsburg, Manos Mavrikakis, and Maria Flytzani-Stephanopoulos (2010) “Alkali-Stabilized Pt-OH_x Species Catalyze Low-Temperature Water-Gas Shift Reactions,” Science 329 (5999), 1633-1636 doi: 10.1126/science.1192449

DOE awarding $4.4M to advance hydropower manufacturing for “low-head” sites

2 December 2014

The US Department of Energy (DOE) is awarding a total of $4.4 million for two projects to support the use of advanced materials and manufacturing techniques in the development of new “low-head” hydropower technologies. Low-head sites, which operate with a change in elevation between 2 and 20 meters, include waterways at existing non-powered dams, canals, and conduits. According to Energy Department assessments, there is a technical resource potential of more than 50 gigawatts of potential capacity at these low-head sites.

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New, low-cost, integrated hydropower turbine and generator sets made with modern materials and manufacturing technologies will help power providers harness the full generating potential of these existing low-head sites and produce cost-competitive, renewable electricity.

• Eaton Corporation will develop a turbine and generator system that uses lightweight advanced materials and advanced manufacturing techniques such as laser-assisted welding, surface treatments, and processing. The turbine will be designed to deliver a constant source of energy despite changes in water flow by using a system that operates efficiently across a range of ebbs and flows. The Eaton Corporation will design, fabricate, and test its turbine at 1/10^th scale.

• Pennsylvania State University will develop and demonstrate a low-head hydropower turbine and generator system prototype that combines lightweight, corrosion-resistant metallic components that can be produced through an additive manufacturing process. A condition-based monitoring system will also facilitate improved operation and maintenance.

Together, these organizations will combine advanced materials and manufacturing techniques to maximize efficiency and improve the design, performance and durability of innovative hydropower manufacturing capabilities.

PNNL team reports growth of dendrite-free lithium films with self-aligned and compact nanorod structure

2 December 2014

Suppressing lithium (Li) dendrite growth is one of the most critical challenges for the development of Li-metal batteries—i.e., high-energy density batteries using a Li-metal anode such as Li-sulfur or Li-air. (Earlier post.) Researchers at Pacific Northwest National Laboratory (PNNL) report for the first time the growth of dendrite-free lithium films with a self-aligned and highly compacted nanorod structure. Their paper appears in the ACS journal Nano Letters.

Lithium metal is a very promising anode material for high-capacity rechargeable batteries due to its theoretical high capacity of 3,860 mAh g⁻¹ (~10x that of the 372 mAh g⁻¹ of graphite anodes in Li-ion batteries), but it fails to meet cycle life and safety requirements due to electrolyte decomposition and dendrite formation on the surfaces of the lithium metal anodes during cycling. Thus, numerous efforts have been and are being made to develop a safe, extended cycling lithium-metal electrode and/or supporting electrolyte (e.g., earlier post, earlier post, earlier post, earlier post.)

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Credit: ACS, Zhang et al. Click to enlarge.

Most of the work in suppression of dendrite formation can be divided into three categories, the team noted in their paper:

• Improve the stability and uniformity of the solid electrolyte interphase (SEI) on the Li electrode surface by optimization of solvents salts, and electrolyte
  additives. However, the SEI alone cannot completely eliminate dendrite formation/growth because of its weak mechanical strength.

• Form alloys of Li and non-Li metal during electrodeposition, which is achieved by adding inorganic compounds or a second salt to the electrolytes. However, most of these metal cation additives will be consumed by forming alloys during Li deposition, so the suppression of Li dendrite formation may not be sustainable.

• Use mechanical barriers to block dendrite growth. However, while such a protective layer acts as an effective physical barrier to block dendrite penetration, it does not alter the growth mechanism of Li dendrites on a fundamental level. As a result, porous Li may still be generated beneath the physical barrier and lead to a rapid increase in the impedance of the Li anode and battery failure even without a dendrite-related short.

The PNNL team itself had earlier developed a cesium hexafluorophosphate (CsPF₆) as an electrolyte additive which was shown to suppress Li dendrite formation.

In this work, we further investigated the evolution of the cross-sectional morphologies of such Li films during deposition/stripping cycles. The effects of Cs⁺ and other components in electrolytes on the voltage profiles of electrochemical deposition and the chemistry of the SEI formed on copper (Cu) substrates prior to Li deposition are systematically investigated. It is revealed for the first time that the apparently dense Li films grown in the presence of CsPF₆ additive actually consist of self-aligned, highly compacted nanorods. We also found that, before the reductive decomposition of carbonate solvents at about 0.9−1.2 V, a thin SEI has already formed on the Cu surface at about 2.05 V vs Li/Li⁺. The quality of this underlying SEI predecessor would essentially dictate the morphologies of the Li to be deposited.

The new understanding on the internal microstructure of the dendrite-free Li films deposited electrochemically, their structural evolution during stripping/deposition processes, and the synergistic effect of Cs⁺ additive and preformed underlying SEI on Li deposition will help researchers in this field design new approaches to enable a metallic Li anode, the “Holy Grail” of Li-based battery chemistries.

Schematic of Li deposition and stripping processes when the Cs additive is used. (a) The initial distribution of cations/anions before Li deposition; (b) redistribution of cations/anions after an electric field is applied and the formation of the initial SEI layer (represented by a blue line) before Li deposition; (c) the initial growth of small Li (represented by gray area) tips; (d) the dendrite−free Li films with self-aligned and highly compact nanorods; (e) Li film after partial stripping; and (f) Li film after redeposition. The blue lines covering at the surface of deposited Li (gray) represent the SEI layer formed during the deposition and stripping processes. Credit: ACS, Zhang et al. Click to enlarge.

Resources

• Yaohui Zhang, Jiangfeng Qian, Wu Xu, Selena M. Russell, Xilin Chen, Eduard Nasybulin, Priyanka Bhattacharya, Mark H. Engelhard, Donghai Mei, Ruiguo Cao, Fei Ding, Arthur V. Cresce, Kang Xu, and Ji-Guang Zhang (2014) “Dendrite-Free Lithium Deposition with Self-Aligned Nanorod Structure” Nano Letters doi: 10.1021/nl5039117

Novozymes launches commercial enzyme technology to convert waste oils into biodiesel

2 December 2014

Novozymes has launched Eversa Transform, the first commercially available enzymatic solution (a liquid lipase) to convert both glycerides and free fatty acids (FFA) into biodiesel. Biodiesel producers can thereby use cooking oil or other lower grade oils as biodiesel feedstock, reducing their raw material costs. The resulting enzymatic biodiesel is sold to the same trade specification as biodiesel created through traditional chemical processing.

Growing demand for vegetable oil in the food industry has resulted in increased prices, causing biodiesel producers to search for alternative—and more sustainable—feedstocks. Most of the oils currently used in biodiesel production are sourced from soybeans, palm or rapeseed, and typically contain less than 0.5% free fatty acids (FFA). Existing biodiesel process designs have difficulty handling oils containing more than 0.5% FFA—i.e., waste oils with high FFAs have not been a viable feedstock option.

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The Eversa Transform reaction is carried out in a mixed batch tank operation. By controlling the reaction conditions, it is possible to ensure a minimum of FFA (typically around 2%) which is eliminated by a polishing step—e.g., a caustic wash step or a resin-catalyzed esterification.

This figure shows the basic layout of the Novozymes enzymatic process. Recommended conditions for the enzyme reactor: 0.7% enzyme, 1.5 equivalent MeOH, 35°C/95°F, 2% water and 20 to 24 hours reaction time. Source: Novozymes. Click to enlarge.

The enzymatic process eliminates the need for sodium methoxide, one of the most hazardous chemicals in traditional biodiesel plants. The significant reduction of harsh chemicals and by-products enhances safety for both personnel and the environment. The Eversa process does not use high pressure or temperature.

Eversa can work with a broad range of fatty materials as feedstock, but initial focus has been on used cooking oil, DDGS corn oil and fatty acid distillates.

The idea of enzymatic biodiesel is not new, but the costs involved have been too high for commercial viability,. Eversa changes this and enables biodiesel producers to finally work with waste oils and enjoy feedstock flexibility to avoid the pinch of volatile pricing.

The enzymatic process uses less energy, and the cost of waste oil as a feedstock is significantly lower than refined oils. A small number of plants have been producing biodiesel from waste oils using existing technologies. But this has not been cost-efficient until now, broadly speaking, as the waste oils have had to be refined before being processed using chemicals. We hope that our technology can unleash more of the potential in these lower grade feedstocks.

Two plants in the US—owned by Blue Sun Energy and Viesel Fuel—are using Novozymes enzymatic solutions for biodiesel production.

The process developed by Blue Sun for enzymatic transesterification improves the bottom line through lower costs and higher revenue.

Making the change from a chemical catalyst to the enzymatic process requires retrofitting in existing plants. Biodiesel producers looking to utilize Eversa will therefore have to invest time and resources to make the switch to the enzymatic process.

Novozymes’ engineering partners estimate that the resulting improved process economy indicates a payback time of three years or less, depending on the plant setup and feedstock savings potential in that region.