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  • richardmitnick 9:57 am on December 3, 2020 Permalink | Reply
    Tags: "Understanding bacteria’s metabolism could improve biofuel production", , , Biofuels, , , , One of the barriers to creating biofuels that are cost competitive with petroleum is the inefficiency of converting plant material into ethanol., The authors describe mathematical and computational modeling; artificial intelligence; algorithms; and experiments showing that cells have failsafe mechanisms.,   

    From UC Riverside: “Understanding bacteria’s metabolism could improve biofuel production” 

    UC Riverside bloc

    From UC Riverside

    December 3, 2020
    Jules Bernstein
    (951) 827-4580


    A new study reveals how bacteria control the chemicals produced from consuming ‘food.’ The insight could lead to organisms that are more efficient at converting plants into biofuels.

    The study, authored by scientists at UC Riverside and Pacific Northwest National Laboratory, has been published in the Journal of the Royal Society Interface.

    Colorized scanning electron micrograph of E. coli, bacteria commonly used in the production of biofuels. Credit: NIAID.

    In the article, the authors describe mathematical and computational modeling, artificial intelligence algorithms and experiments showing that cells have failsafe mechanisms preventing them from producing too many metabolic intermediates.

    Metabolic intermediates are the chemicals that couple each reaction to one another in metabolism. Key to these control mechanisms are enzymes, which speed up chemical reactions involved in biological functions like growth and energy production.

    “Cellular metabolism consists of a bunch of enzymes. When the cell encounters food, an enzyme breaks it down into a molecule that can be used by the next enzyme and the next, ultimately generating energy,” explained study co-author, UCR adjunct math professor and Pacific Northwest National Laboratory computational scientist William Cannon.

    The enzymes cannot produce an excessive amount of metabolic intermediates. They produce an amount that is controlled by how much of that product is already present in the cell.

    “This way the metabolite concentrations don’t get so high that the liquid inside the cell becomes thick and gooey like molasses, which could cause cell death,” Cannon said.

    One of the barriers to creating biofuels that are cost competitive with petroleum is the inefficiency of converting plant material into ethanol. Typically, E. coli bacteria are engineered to break down lignin, the tough part of plant cell walls, so it can be fermented into fuel.

    Mark Alber, study co-author and UCR distinguished math professor, said that the study is a part of the project to understand the ways bacteria and fungi work together to affect the roots of plants grown for biofuels.

    “One of the problems with engineering bacteria for biofuels is that most of the time the process just makes the bacteria sick,” Cannon said. “We push them to overproduce proteins, and it becomes uncomfortable — they could die. What we learned in this research could help us engineer them more intelligently.”

    Knowing which enzymes need to be prevented from overproducing can help scientists design cells that produce more of what they want and less of what they don’t.

    The research employed mathematical control theory, which learns how systems control themselves, as well as machine learning to predict which enzymes needed to be controlled to prevent excessive buildup of metabolites.

    While this study examined central metabolism, which generates the cell’s energy, going forward, Cannon said the research team would like to study other aspects of a cell’s metabolism, including secondary metabolism — how proteins and DNA are made — and interactions between cells.

    “I’ve worked in a lab that did this kind of thing manually, and it took months to understand how one particular enzyme is regulated,” Cannon said. “Now, using these new methods, this can be done in a few days, which is extremely exciting.”

    The U.S. Department of Energy, seeking to diversify the nation’s energy sources, funded this three-year research project with a $2.1 million grant.

    The project is also a part of the broader initiatives under way in the newly established UCR Interdisciplinary Center for Quantitative Modeling in Biology.

    Though this project focused on bacterial metabolism, the ability to learn how cells regulate and control themselves could also help develop new strategies for combatting diseases.

    “We’re focused on bacteria, but these same biological mechanisms and modeling methods apply to human cells that have become dysregulated, which is what happens when a person has cancer,” Alber said. “If we really want to understand why a cell behaves the way it does, we have to understand this regulation.”

    See the full article here .


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  • richardmitnick 1:01 pm on November 14, 2017 Permalink | Reply
    Tags: Advanced Biofuels and Bioproducts Process Demonstration Unit (ABPDU), , , Biofuels, , Here we’re cultivating an entire community of microbes to access enzymes that we couldn’t get from isolates, , , Metagenomic analysis, New types of cellulases enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts   

    From LBNL: “To Find New Biofuel Enzymes, It Can Take a Microbial Village” 

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    Berkeley Lab

    November 14, 2017
    Sarah Yang
    (510) 486-4575

    A new study led by researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory (Berkeley Lab), demonstrates the importance of microbial communities as a source of stable enzymes that could be used to convert plants to biofuels.

    This 50-milliliter flask contains a symbiotic mix of bacteria derived from compost that was maintained for three years. (Credit: Steve Singer/JBEI)

    The study, recently published in the journal Nature Microbiology, reports on the discovery of new types of cellulases, enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts. The cellulases were cultured from a microbiome. Using a microbial community veers from the approach typically taken of using isolated organisms to obtain enzymes.

    The scientists first studied the microbial menagerie present in a few cups of municipal compost. Metagenomic analysis at the DOE Joint Genome Institute (JGI) of the microbiome helped reveal that 70 percent of the enzymatic activity originated from cellulases produced by a cluster of uncultivated bacteria in the compost. They found that the enzymes easily broke down the cellulose in plant biomass into glucose at temperatures up to 80 degrees Celsius.

    This chart shows the bacterial composition of the community in the bioreactor after two weeks of culturing. (Credit: Sebastian Kolinko/JBEI)

    “Here we’re cultivating an entire community of microbes to access enzymes that we couldn’t get from isolates,” said study principal investigator Steve Singer, senior scientist in Berkeley Lab’s Biological Systems and Engineering Division and director of Microbial and Enzyme Discovery at JBEI. “Some microbes are difficult to culture in a lab. We are cultivating microbes living in communities, as they occur in the wild, which allows us to see things we don’t see when they are isolated. This opens up the opportunity to discover new types of enzymes that are only produced by microbes in communities.”

    The bacterial population, Candidatus Reconcilibacillus cellulovorans, yielded cellulases that were arranged in remarkably robust carbohydrate-protein complexes, a structure never before observed in isolates. The stability of the new cellulase complexes makes them attractive for applications in biofuels production, the study authors said.

    “The enzymes persist, even after a decline in bacterial abundance,” said Singer, who compared the microbial community with sourdough starters fermented from wild yeast and friendly bacteria. “We kept the microbial community cultivation going for more than three years in the lab.”

    A bioreactor at ABPDU was used to scale the growth of a mixture of bacteria from 50 milliliters to 300 liters. (Credit: Roy Kaltschmidt/Berkeley Lab).

    This stability is a key advantage over other cellulases that degrade more rapidly at high temperature, the researchers said.

    To determine whether the enzyme production can be scalable for industrial applications, JBEI scientists collaborated with researchers from the Advanced Biofuels and Bioproducts Process Demonstration Unit (ABPDU) at Berkeley Lab, a scale-up facility established by DOE to help accelerate the commercialization of biofuels research discoveries.

    Researchers at JBEI, a DOE Bioenergy Research Center, were able to produce 50-milliliter samples, but in about six weeks, the scientists at ABPDU scaled the cultures to a volume 6,000 times larger – 300 liters – in industrial bioreactors.

    The study’s lead author is Sebastian Kolinko, who worked on the study as a JBEI postdoctoral researcher.

    Other co-authors on this study include researchers from Taipei Medical University, the University of Georgia, the Manheim University of Applied Sciences, and Technical University of Braunschweig in Germany.

    JGI is a DOE Office of Science User Facility. This work was primarily supported by the DOE Office of Science and the DOE Office of Energy Efficiency and Renewable Energy.

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  • richardmitnick 8:04 am on July 7, 2017 Permalink | Reply
    Tags: , Biofuels, ,   

    From Rutgers: “Cutting the Cost of Ethanol, Other Biofuels and Gasoline” 

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    Rutgers University

    July 5, 2017
    Todd B. Bates

    Enzymes, genetically engineered to avoid sticking to the surfaces of biomass such as corn stalks, may lower costs in the production of cellulose-based biofuels like ethanol. Shishir Chundawat/Rutgers University and U.S. Department of Energy

    Biofuels like the ethanol in U.S. gasoline could get cheaper thanks to experts at Rutgers University-New Brunswick and Michigan State University.

    They’ve demonstrated how to design and genetically engineer enzyme surfaces so they bind less to corn stalks and other cellulosic biomass, reducing enzyme costs in biofuels production, according to a study published this month on the cover of the journal ACS Sustainable Chemistry & Engineering.

    “The bottom line is we can cut down the cost of converting biomass into biofuels,” said Shishir P. S. Chundawat, senior author of the study and an assistant professor in the Department of Chemical and Biochemical Engineering at Rutgers University-New Brunswick.

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  • richardmitnick 3:19 pm on June 28, 2017 Permalink | Reply
    Tags: Biofuels, IMAGINE neutron scattering diffractometer, LPMOs - lytic polysaccharide monooxygenases, , , , ORNL’s High Flux Isotope Reactor   

    From ORNL: “‘On your mark, get set'” 


    Oak Ridge National Laboratory

    June 27, 2017
    Jeremy Rumsey

    A combination of X-ray and neutron scattering has revealed new insights into how a highly efficient industrial enzyme is used to break down cellulose. Knowing how oxygen molecules (red) bind to catalytic elements (illustrated by a single copper ion) will guide researchers in developing more efficient, cost-effective biofuel production methods. (Image credit: ORNL/Jill Hemman)

    Producing biofuels like ethanol from plant materials requires various enzymes to break down the cellulosic fibers. Scientists using neutron scattering have identified the specifics of an enzyme-catalyzed reaction that could significantly reduce the total amount of enzymes used, improving production processes and lowering costs.

    Researchers from the Department of Energy’s Oak Ridge National Laboratory and North Carolina State University used a combination of X-ray and neutron crystallography to determine the detailed atomic structure of a specialized fungal enzyme.

    A deeper understanding of the enzyme reactivity could also lead to improved computational models that will further guide industrial applications for cleaner forms of energy. Their results are published in the journal Angewandte Chemie International Edition.

    Part of a larger family known as lytic polysaccharide monooxygenases, or LPMOs, these oxygen-dependent enzymes act in tandem with hydrolytic enzymes—which chemically break down large complex molecules with water—by oxidizing and breaking the bonds that hold cellulose chains together. The combined enzymes can digest biomass more quickly than currently used enzymes and speed up the biofuel production process.

    “These enzymes are already used in industrial applications, but they’re not well understood,” said lead author Brad O’Dell, a graduate student from NC State working in the Biology and Soft Matter Division of ORNL’s Neutron Sciences Directorate. “Understanding each step in the LPMO mechanism of action will help industry use these enzymes to their full potential and, as a result, make final products cheaper.”

    In an LPMO enzyme, oxygen and cellulose arrange themselves through a sequence of steps before the biomass deconstruction reaction occurs. Sort of like “on your mark, get set, go,” says O’Dell.

    To better understand the enzyme’s reaction mechanism, O’Dell and coauthor Flora Meilleur, ORNL instrument scientist and an associate professor at NC State, used the IMAGINE neutron scattering diffractometer at ORNL’s High Flux Isotope Reactor to see how the enzyme and oxygen molecules were behaving in the steps leading up to the reaction—from the “resting state” to the “active state.”

    ORNL IMAGINE neutron scattering diffractometer

    The resting state, O’Dell says, is where all the critical components of the enzyme assemble to bind oxygen and carbohydrate. When electrons are delivered to the enzyme, the system moves from the resting state to the active state—i.e., from “on your mark” to “get set.”

    In the active state, oxygen binds to a copper ion that initiates the reaction. Aided by X-ray and neutron diffraction, O’Dell and Meilleur identified a previously unseen oxygen molecule being stabilized by an amino acid, histidine 157.

    Hydrogen is a key element of amino acids like histidine 157. Because neutrons are particularly sensitive to hydrogen atoms, the team was able to determine that histidine 157 plays a significant role in transporting oxygen molecules to the copper ion in the active site, revealing a vital detail about the first step of the LPMO catalytic reaction.

    “Because neutrons allow us to see hydrogen atoms inside the enzyme, we gained essential information in deciphering the protein chemistry. Without that data, the role of histidine 157 would have remained unclear,” Meilleur said. “Neutrons were instrumental in determining how histidine 157 stabilizes oxygen to initiate the first step of the LPMO reaction mechanism.”

    Their results were subsequently confirmed via quantum chemical calculations performed by coauthor Pratul Agarwal from ORNL’s Computing and Computational Sciences Directorate.

    Research material preparation was supported by the ORNL Center for Structural Molecular Biology. X-ray data were collected at the Argonne National Laboratory Advanced Photon Source through access provided by the Southeast Regional Collaborative Access Team.

    O’Dell says their results refine the current understanding of LPMOs for science and industry researchers.

    “This is a big step forward in unraveling how LPMO’s initiate the breakdown of carbohydrates,” O’Dell said. “Now we need to characterize the enzyme’s activated state when the protein is also bound to a carbohydrate that mimics cellulose. Then we’ll have the chance to see what structural changes happen when the starting pistol is fired and the reaction takes off.”

    HFIR is a DOE Office of Science User Facility. UT-Battelle manages ORNL for the Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, please visit http://science.energy.gov/.

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  • richardmitnick 11:56 am on June 20, 2017 Permalink | Reply
    Tags: , Biofuels, , , ,   

    From COSMOS: “Precision gene editing may deliver biofuel promise” 

    Cosmos Magazine bloc


    20 June 2017
    Elizabeth Finkel

    Biofuels from algae come with great promise. Santiago Urquijo/Getty Images

    For decades now, we’ve been promised cheap biofuels from algae. But there’s no free lunch. Growing these mini oil factories in vast ponds requires fertiliser and mechanical aeration; and then the oil has to be extracted. It all costs energy and money so the yields need to be high to make it worthwhile.

    One promising industrial species is Nannocholoropsis gaditana, which can produce a lipid – the oil and fat energy store – content up to about 60% of the algae’s ash-free dry weight. But Eric Moellering and colleagues at the company Synthetic Genomics Inc in California, wanted to do better.

    Starving the algae of nitrogen, paradoxically, boosts oil production. The problem is that the plants also curtail their growth so there’s no net gain. Ever since the 1970s, scientists have been trying to genetically engineer their way out of this quandary. But they’ve had to wait for the right tool: the bacterially derived CRISPR–Cas9 enzyme that has transformed ham-fisted genetic engineering into precision gene-editing.

    Moellering’s group identified a gene, called ZnCys, that was deactivated when the algae was starved of nitrogen. When the researchers completely disabled that gene, they saw oil production double, even without starving the algae.

    But the algae still grew poorly, so the scientists made use of the finesse of CRISPR–Cas9 to finely edit the DNA code of the ZnCys gene instead of disabling it competely. As they report in Nature Biotechnology this led to doubling oil yield without dampening algae growth.

    The final yield was up to five grams per square metre of algae per day.

    Amazing what a good editor can do!

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  • richardmitnick 5:53 am on May 23, 2017 Permalink | Reply
    Tags: , Biofuels, Discovery of an alga’s ‘dictionary of genes’ could lead to advances in biofuels and medicine, ,   

    From UCLA: “Discovery of an alga’s ‘dictionary of genes’ could lead to advances in biofuels, medicine” 

    UCLA bloc


    May 22, 2017
    Stuart Wolpert

    Inside the alga’s cells, showing the nucleus (purple), mitochondria (red), chloroplast (green) and lipids (yellow). Melissa Roth/HHMI and Andreas Walters/Berkeley Lab

    Plant biologists and biochemists from UCLA, UC Berkeley and UC San Francisco have produced a gold mine of data by sequencing the genome of a green alga called Chromochloris zofingiensis.


    Scientists have learned in the past decade that the tiny, single-celled organism could be used as a source of sustainable biofuel and that it produces a substance called astaxanthin, which may be useful for treating certain diseases. The new research could be an important step toward improving production of astaxanthin by algae and engineering its production in plants and other organisms.

    The study is published online in the journal Proceedings of the National Academy of Sciences.

    Chromochloris zofingiensis is one of the most prolific producers of a type of lipids called triacylglycerols, which are used in producing biofuels.

    Knowing the genome is like having a “dictionary” of the alga’s approximately 15,000 genes, said co-senior author Sabeeha Merchant, a UCLA professor of biochemistry. “From there, researchers can learn how to put the ‘words’ and ‘sentences’ together, and to target our research on important subsets of genes.”

    C. zofingiensis provides an abundant natural source for astaxanthin, an antioxidant found in salmon and other types of fish, as well as in some birds’ feathers. And because of its anti-inflammatory properties, scientists believe astaxanthin may have benefits for human health; it is being tested in treatments for cancer, cardiovascular disease, neurodegenerative diseases, inflammatory diseases, diabetes and obesity. Merchant said the natural version has stronger antioxidant properties than chemically produced ones, and only natural astaxanthin has been approved for human consumption.

    The study also revealed that an enzyme called beta-ketolase is a critical component in the production of astaxanthin.

    Algae absorb carbon dioxide and derive their energy from sunlight, and C. zofingiensis in particular can be cultivated on non-arable land and in wastewater. Harnessing it as a source for renewable and sustainable biofuels could lead to new ways to produce clean energy, said Krishna Niyogi, co-senior author of the paper and a scientist at the Department of Energy’s Lawrence Berkeley National Laboratory.

    Over the past decade-plus, Merchant said, research with algae, a small plant called rockcress, fruit flies and nematode worms — all so-called “model organisms” — has been advanced by other scientists’ determining their genome sequences.

    “They are called model organisms because we use what we learn about the operation of their cells and proteins as a model for understanding the workings of more complex systems like humans or crops,” she said. “Today, we can sequence the genome of virtually any organism in the laboratory, as has been done over the past 10 to 15 years with other model organisms.”

    Merchant, Niyogi and Matteo Pellegrini, a UCLA professor of molecular, cell and developmental biology and a co-author of the study, maintain a website that shares a wealth of information about the alga’s genome.

    During the study, the scientists also used soft X-ray tomography, a technique similar to a CT scan, to get a 3-D view of the algae cells , which gave them more detailed insights about their biology.

    Niyogi is also a UC Berkeley professor of plant and microbial biology and a Howard Hughes Medical Institute Investigator.


    The study’s other authors are researchers Shawn Cokus and Sean Gallaher and postdoctoral scholar David Lopez, all of UCLA; postdoctoral fellow Melissa Roth, and graduate students Erika Erickson, Benjamin Endelman and Daniel Westcott, all of Niyogi’s laboratory; and Carolyn Larabell, a professor of anatomy, and researcher Andreas Walter, both of UC San Francisco.

    The research was funded by the Department of Energy’s Office of Science, the Department of Agriculture’s National Institute of Food and Agriculture, the National Institute of General Medical Sciences of the National Institutes of Health, and the Gordon and Betty Moore Foundation.

    See the full article here .

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  • richardmitnick 12:26 pm on August 8, 2016 Permalink | Reply
    Tags: , Biofuels, ,   

    From MITEI at MIT: “Microbial engineering technique could reduce contamination in biofermentation plants” 

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    August 4, 2016
    Helen Knight

    Approach could lower cost and eliminate need for antibiotics during biofuel production.

    The ability to ferment low-cost feedstocks under nonsterile conditions may enable new classes of biochemicals and biofuels, such as microbial oil produced by the yeast Yarrowia lipolytica (shown here, oil in lipid bodies is stained green and cells walls stained blue). Photo: Novogy, Inc.

    The cost and environmental impact of producing liquid biofuels and biochemicals as alternatives to petroleum-based products could be significantly reduced, thanks to a new metabolic engineering technique.

    Liquid biofuels are increasingly used around the world, either as a direct “drop-in” replacement for gasoline, or as an additive that helps reduce carbon emissions.

    The fuels and chemicals are often produced using microbes to convert sugars from corn, sugar cane, or cellulosic plant mass into products such as ethanol and other chemicals, by fermentation. However, this process can be expensive, and developers have struggled to cost-effectively ramp up production of advanced biofuels to large-scale manufacturing levels.

    One particular problem facing producers is the contamination of fermentation vessels with other, unwanted microbes. These invaders can outcompete the producer microbes for nutrients, reducing yield and productivity.

    Ethanol is known to be toxic to most microorganisms other than the yeast used to produce it,Saccharomyces cerevisiae, naturally preventing contamination of the fermentation process. However, this is not the case for the more advanced biofuels and biochemicals under development.

    To kill off invading microbes, companies must instead use either steam sterilization, which requires fermentation vessels to be built from expensive stainless steels, or costly antibiotics. Exposing large numbers of bacteria to these drugs encourages the appearance of tolerant bacterial strains, which can contribute to the growing global problem of antibiotic resistance.

    Now, in a paper published today in the journal Science, researchers at MIT and the Cambridge startup Novogy describe a new technique that gives producer microbes the upper hand against unwanted invaders, eliminating the need for such expensive and potentially harmful sterilization methods.

    The researchers engineered microbes, such as Escherichia coli, with the ability to extract nitrogen and phosphorous — two vital nutrients needed for growth — from unconventional sources that could be added to the fermentation vessels, according to Gregory Stephanopoulos, the Willard Henry Dow Professor of Chemical Engineering and Biotechnology at MIT, and Joe Shaw, senior director of research and development at Novogy, who led the research.

    What’s more, because the engineered strains only possess this advantage when they are fed these unconventional chemicals, the chances of them escaping and growing in an uncontrolled manner outside of the plant in a natural environment are extremely low.

    “We created microbes that can utilize some xenobiotic compounds that contain nitrogen, such as melamine,” Stephanopoulos says. Melamine is a xenobiotic, or artificial, chemical that contains 67 percent nitrogen by weight.

    Conventional biofermentation refineries typically use ammonium to supply microbes with a source of nitrogen. But contaminating organisms, such as Lactobacilli, can also extract nitrogen from ammonium, allowing them to grow and compete with the producer microorganisms.

    In contrast, these organisms do not have the genetic pathways needed to utilize melamine as a nitrogen source, says Stephanopoulos.

    “They need that special pathway to be able to utilize melamine, and if they don’t have it they cannot incorporate nitrogen, so they cannot grow,” he says.

    The researchers engineered E. coli with a synthetic six-step pathway that allows it to express enzymes needed to convert melamine to ammonia and carbon dioxide, in a strategy they have dubbed ROBUST (Robust Operation By Utilization of Substrate Technology).

    When they experimented with a mixed culture of the engineered E. coli strain and a naturally occurring strain, they found the engineered type rapidly outcompeted the control, when fed on melamine.

    They then investigated engineering the yeast Saccharomyces cerevisiae to express a gene that allowed it to convert the nitrile-containing chemical cyanamide into urea, from which it could obtain nitrogen.

    The engineered strain was then able to grow with cyanamide as its only nitrogen source.

    Finally, the researchers engineered both S. cerevisiae and the yeast Yarrowia lipolytica to use potassium phosphite as a source of phosphorous.

    Like the engineered E. coli strain, both the engineered yeasts were able to outcompete naturally occurring strains when fed on these chemicals.

    “So by engineering the strains to make them capable of utilizing these unconventional sources of phosphorous and nitrogen, we give them an advantage that allows them to outcompete any other microbes that may invade the fermenter without sterilization,” Stephanopoulos says.

    The microbes were tested successfully on a variety of biomass feedstocks, including corn mash, cellulosic hydrolysate, and sugar cane, where they demonstrated no loss of productivity when compared to naturally occurring strains.

    The paper provides a novel approach to allow companies to select for their productive microbes and select against contaminants, according to Jeff Lievense, a senior engineering fellow at the San Diego-based biotechnology company Genomatica who was not involved in the research.

    “In theory you could operate a fermentation plant with much less expensive equipment and lower associated operating costs,” Lievense says. “I would say you could cut the capital and capital-related costs [of fermentation] in half, and for very large-volume chemicals, that kind of saving is very significant,” he says.

    The ROBUST strategy is now ready for industrial evaluation, Shaw says. The technique was developed with Novogy researchers, who have tested the engineered strains at laboratory scale and trials with 1,000-liter fermentation vessels, and with Felix Lam of the MIT Whitehead Institute for Biomedical Research, who led the cellulosic hydrosylate testing.

    Novogy now hopes to use the technology in its own advanced biofuel and biochemical production, and is also interested in licensing it for use by other manufacturers, Shaw says.

    See the full article here .

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  • richardmitnick 8:36 am on July 1, 2016 Permalink | Reply
    Tags: , Biofuels,   

    From BNL: “Study Shows Trees with Altered Lignin are Better for Biofuels” 

    Brookhaven Lab

    June 28, 2016
    Karen McNulty Walsh
    (631) 344-8350

    Peter Genzer
    (631) 344-3174

    Fundamental enzyme study leads to increased access to bioenergy feedstocks and improves ethanol yield by modifying plant cell wall structures.

    Postdoctoral associate Yuanheng Cai, biological research associate Xuebin Zhang, and plant biochemist Chang-Jun Liu in the Brookhaven Lab greenhouse with transgenic trees designed to improve biofuel production.

    Lignin is a natural component of plant cell walls, the scaffolding that surrounds each cell and plays a pivotal role in plants’ ability to grow against gravity and reach heights ranging from stubbly grasses to the sky-scraping splendor of redwoods. But lignin is a problem for scientists interested in converting plant biomass to biofuels and other sustainable bio-based products. Lignin makes it hard to break down the plant matter so its carbon-rich building blocks can be converted into forms suitable for generating energy or running automobiles.

    A simple solution might be to engineer plants with less lignin. But previous attempts to do this have often resulted in weaker plants and stunted growth—essentially putting the brakes on biomass production.

    Now, by engineering a novel enzyme involved in lignin synthesis, scientists at the U.S. Department of Energy’s Brookhaven National Laboratory and collaborators have altered the lignin in aspen trees in a way that increases access to biofuel building blocks without inhibiting plant growth. Their research, described in Nature Communications, resulted in an almost 50 percent increase in ethanol yield from healthy aspen trees whose woody biomass released 62 percent more simple sugars than native plants.

    “Our study provides a useful strategy for tailoring woody biomass for bio-based applications,” said Brookhaven biologist Chang-Jun Liu, the lead author on the project.

    Lignin makes up about 20 percent of aspen’s woody structures, with cellulose and hemicellulose polymers making up approximately 45 and 25 percent, along with other minor components.

    “The lignin forms a barrier of sorts around the other polymers,” Liu explained. “Digestive enzymes can’t get through to break down the cellulose and hemicellulose to release their simple sugars.”

    Prior work, including Liu’s own research manipulating enzymes involved in lignin synthesis, has shown that reducing or altering plants’ lignin content can make woody biomass more digestible. But many of these approaches, particularly those that dramatically reduced lignin content, resulted in weaker plants and severe reductions in biomass yield, rendering these plants unsuitable for large-scale cultivation.

    In this study the scientists explored a creative new strategy for modifying lignin’s structure based on detailed analysis of enzyme structures that were previously solved by Liu’s group using x-rays at the National Synchrotron Light Source (NSLS)—a DOE Office of Science User Facility at Brookhaven Lab, now replaced by a much brighter NSLS-II.

    BNL NSLS Interior

    BNL NSLS-II Interior

    That work, described in papers published in Plant Cell (2012) and the Journal of Biological Chemistry (2010 and 2015), was part of an effort to understand the enzymes’ mechanism of selectivity. In those studies, the scientists also sought to engineer a series of variations of the enzyme, called monolignol 4-O-methyltransferase, some of which effectively modified the structure of lignin building blocks so they would no longer be incorporated into the lignin polymer.

    Cross-sections of wild type (a,c) and transgenic (b,d) aspen stems stained with lignin detection agents. The violet red color in the upper images indicates no dramatic change in total lignin content between the wild type (a) and transgenic (b) plants. The red color staining specific for one particular lignin structural unit shown in the lower images, however, indicates a significantly lower level of this component in the transgenic aspen (d) compared with the wild type (c). These findings suggest that lignin in the transgenic aspen has significantly altered composition and structure.

    In the new work, the scientists used biochemical analyses to identify a variant of monolignol 4-O-methyltransferase that had a slight chemical “preference” for reacting with one specific type of lignin precursor. The scientists reasoned that this variant had the potential to depress the formation of a particular lignin component.

    To test this idea, they transplanted the gene for this variant into a strain of fast-growing aspen trees—a model for other trees in the poplar family, which have widespread potential for bioenergy production because of their ability to grow in many regions and on non-agricultural land. The scientists grew the altered aspen trees alongside untreated control trees in a greenhouse on Brookhaven’s property.

    Modified cell walls, more sugar

    The trees that produced the engineered enzyme had slightly less total lignin in their cell walls. But on further analysis, the scientists found that these trees also had dramatically altered lignin structure, with a significant reduction in the level of one of the two major types of lignin components normally found in aspen trees. These findings were further confirmed using two-dimensional nuclear magnetic resonance spectroscopic imaging by a team led by John Ralph of the University of Wisconsin and the Great Lakes Bioenergy Research Center, a DOE Bioenergy Research Center. Specifically, the engineered trees had less “labile” lignin, while the remaining lignin components became structurally more condensed, forming an increased number of cross-linkages among the polymers.

    “We expected that this condensed, more cross-linked lignin might make the plants even harder to digest, but found that wood containing these structures released up to 62 percent more simple sugars when treated with digestive enzymes,” Liu said. The yield of ethanol from this modified wood was almost 50 percent higher than the ethanol yield of wood derived from untreated control trees.

    Microscopic images of wood tissues from wild type (a) and transgenic (b) aspen trees show similar anatomical and structural features, suggesting that the transgenic plant maintains normal growth and wood formation even with altered lignin composition.

    Interestingly, by imaging aspen wood samples using infrared light at NSLS, the scientists found that their approach for altering lignin content and composition also increased the production of cellulose fibers, the major source of fermentable sugars in the cell wall. This increased cellulose content might partially contribute to the increased release of simple sugars, they said.

    Importantly, the changes in lignin and cell wall structures did not affect the growth of the engineered aspens. The wood densities and the biomass yields were comparable to those of the control trees.

    “These data suggest that lignin condensation itself is not a critical factor affecting the digestibility of the cell wall,” said Liu. “The findings also support the idea that engineering the enzymes that modify lignin precursors represents a useful biotechnological solution for effectively tailoring the digestibility of poplar-family woody biomass to generate feedstocks for biofuel production.

    “It’s gratifying when fundamental studies of enzyme function, such as the findings that underpin this work, can be translated to contribute to solving real-world problems,” he added.

    This work was supported by the DOE Office of Science.

    See the full article here .

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    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.

  • richardmitnick 11:32 am on June 9, 2016 Permalink | Reply
    Tags: , Biofuels, Chemistry lessons from bacteria may improve biofuel production,   

    From Wisconsin: “Chemistry lessons from bacteria may improve biofuel production” 

    U Wisconsin

    University of Wisconsin

    June 8, 2016
    Chris Barncard

    If you’re made of carbon, precious few things are as important to life as death.

    A dead tree may represent a literal windfall of the building blocks necessary for making new plants and animals and the energy to sustain them.

    “The recycling of plant carbon is fundamental to the function of our ecosystems,” says Cameron Currie, professor of bacteriology at the University of Wisconsin–Madison. “We get food, water, air, energy — almost everything — through those ecosystem services. It’s how our planet operates.”

    But the component parts of a dead tree were carefully assembled in the first place, and don’t just fall apart for easy recycling.

    The ability to break down the cellulose in plant material is rare in Streptomyces bacteria, except in strains that live alongside insects — honeybees, leaf-cutter ants (above), some beetles —that eat or make use of the woody parts of plants. Courtesy of Don Parsons/UW-Madison

    In the case of cellulose — a key structural component in plant cell walls and the most abundant organic compound in life on land — a world of specialized microbes handles this careful deconstruction. Much of that work is done by fungi growing on decaying plants, but bacteria in the soil, in the guts of animals like cows and working alongside insects, get the job done, too.

    A new analysis of a group of bacteria called Streptomyces reveals the way some strains of the microbe developed advanced abilities to tear up cellulose, and points out more efficient ways we might mimic those abilities to make fuel from otherwise unusable plant material.

    Streptomyces were long thought to be prominent contributors at work in breaking down cellulose, and to be equally active in the cause across hundreds or thousands of strains of the bacteria.

    Researchers tried to grow different strains of Streptomyces bacteria on dead plant material (in this case, filter paper). Successful cellulose processing strains tapped special genes to produce enzymes that break down cellulose. Courtesy of Currie Lab/UW-Madison

    “The strains that aren’t good at degrading cellulose mostly express the same genes whether we grow them on glucose or on plant material,” says Gina Lewin, a bacteriology graduate student and study co-author. “The strains that are really good at degrading cellulose totally change their gene expression when we grow them on plant material.”

    The successful Streptomyces strains — which were typically those found living in communities with insects — ramp up production of certain enzymes, the proteins that do the cleaving and dissolving and picking apart of cellulose.

    “There are families — six or eight or maybe 10 of these enzymes — that all of the active Streptomyces have,” Fox says. “And this paper shows that the most abundant one of them has to be there or the whole thing collapses.”

    It’s the particular combinations of enzymes that makes the research useful to scientists working on biofuels.

    Counter to long-held belief about the bacteria Streptomyces, seen growing here in a petri dish, the ability to break down a stubborn molecule in plant cell walls called cellulose may be limited to just a few gifted strains. Courtesy of Adam Book/UW-Madison

    Biofuels are typically made from the sugars easily extracted from the same parts of plants we eat.

    “We eat the kernels off the corn cob,” Currie says. “But most of the energy in that corn plant is in the part which is not digestible to us. It’s not in the cob. It’s in the green parts, like the stalk and the leaves.”

    Evolving microbes like Streptomyces have been sharpening the way they make use of those parts of plants almost as long as the plants themselves have been growing on land. That’s hundreds of millions of years. On the other hand, the Department of Energy’s UW–Madison-based Great Lakes Bioenergy Research Center, which funded the Streptomyces study, was established in 2007.

    “The natural world is responding to the same kind of things that humans are,” Fox says. “We need to get food. We need to get energy. And different types of organisms are achieving their needs in different ways. It’s worth looking at how they do it.”

    The new study identifies important enzymes, and new groups of enzymes, produced when Streptomyces flex particular genes. If they represent an improvement over current industrial processes, the microbes’ tricks could make for a great boon to bioenergy production.

    “For a cellulosic biofuel plant, enzymes are one of the most expensive parts of making biofuels,” Lewin says. “So, if you can identify enzymes that work even just slightly better, that could mean a difference of millions of dollars in costs and cheaper energy.”

    See the full article here .

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    In achievement and prestige, the University of Wisconsin–Madison has long been recognized as one of America’s great universities. A public, land-grant institution, UW–Madison offers a complete spectrum of liberal arts studies, professional programs and student activities. Spanning 936 acres along the southern shore of Lake Mendota, the campus is located in the city of Madison.

  • richardmitnick 7:40 am on March 31, 2016 Permalink | Reply
    Tags: , Biofuels, Ee-Been Goh, , woman scientist   

    From LBL: “From Near-Dropout to PhD, Berkeley Lab Scientist Now at Forefront of Biofuels Revolution” 

    Berkeley Logo

    Berkeley Lab

    March 30, 2016
    Julie Chao
    (510) 486-6491

    Ee-Been Goh works with summer intern Joshua Borrajo in 2013. (Credit: Roy Kaltschmidt/Berkeley Lab)

    To see biochemist Ee-Been Goh in the lab today, figuring out how to rewire bacteria to produce biofuels, one would never guess she was once so uninterested in school that she barely made it through junior high.

    “My mom would say, ‘You used to be the worst in school—why would you want to go for more school?’” Goh said. “That was the joke when I was going into grad school.”

    Today Goh is a project scientist at the Joint BioEnergy Institute (JBEI), a Department of Energy Bioenergy Research Center led by Lawrence Berkeley National Laboratory (Berkeley Lab). She has been lead author on two important publications on methyl ketones, a compound found in blue cheese that has also turned out be one of the most promising and high-performing biofuels at JBEI.

    Her path into science is an object lesson she likes to share with the high school students she mentors through a JBEI summer program called Introductory College Level Experience in Microbiology (iCLEM). “I always tell the iCLEM kids how I was a terrible student in Singapore,” she said. “I was a tomboy, and I just loved goofing off on the streets and playing sports. I really hated school. I would’ve loved to have had a career in professional sports.”

    At the age of 14, her parents moved the entire family to Vancouver, largely for a better educational experience for Goh and her two sisters. Getting to do hands-on science activities in high school, such as animal dissections, sparked her interest in science. “When you do it yourself, you understand things a lot better than when you’re being bombarded with words on a board or an overhead projector,” she said. “That’s something that really caught on for me.”

    Likewise, she encourages students to take advantage of any opportunity they might have. “I think some of these students get really dejected when they don’t do well in school, and as you get more dejected, you just don’t want to put in any more effort,” she said. “That’s how I felt when I was a little kid. The most important thing for me is to fan their interest.”

    As an undergrad at the University of British Columbia, her opportunity was an internship in the microbiology lab of Julian Davies, a renowned scientist in the field of antibiotics and drug resistance. “He was retired and in his 70s, but he really loved science so he was still taking on undergrad students who liked to do research,” she said. “He was very hands-on and got very excited even with small results, and it got me excited too.”

    With encouragement from Davies, Goh decided to pursue an advanced career in science. She did her graduate work in microbiology at UC Davis, where her research focused on understanding how bacteria communicate with each other. This work in the basic sciences taught Goh useful research techniques, but she realized she wanted to pursue a career in the applied sciences.

    “With applied science it impacts more people and has broader reach,” she said. “People can take it and use it. That’s what science is about, right?”

    Landing a job at JBEI fit that requirement perfectly.

    From blue cheese to climate change

    “Many valuable commodity chemicals and fragrances are often derived from petroleum,” Goh said. “At JBEI we’re aiming not just to make biofuels but also to replace a lot of everyday products that come from that barrel of crude oil. We’re trying to do our part in slowing down climate change—that’s the ultimate goal.”

    The focus of her research has been methyl ketones, which are derived from fatty acids and show great promise for biodiesel fuels. Not only are methyl ketones a potential biodiesel, there is a market for these compounds as flavors and fragrances. Working with Harry Beller, director of Biofuels Pathways at JBEI, Goh’s research is aimed at engineering E. coli bacteria to produce methyl ketones as efficiently as possible.

    “Ee-Been has made significant scientific contributions to the development of new biofuels at JBEI,” Beller said. “Her two first-author publications on engineering and optimizing a novel methyl ketone pathway in E. coli have made a notable impact in the area of fatty acid-derived biofuels—medium-chain methyl ketones are among our best performing biofuels at JBEI.”

    Beller elaborated on what made Goh good at what she does: “Qualities that have helped Ee-Been be a successful scientist are her persistence and intelligent approach to troubleshooting. In biotechnology, as in many areas of science and technology, only a fraction of scientific avenues we follow lead to clear success. Ee-Been has the fortitude and ability to learn from things that didn’t work to eventually pursue things that do.”

    And although she occasionally likes to pull pranks in the lab—switching out medium-sized gloves for small ones is a perennial favorite—her JBEI colleagues have voted her “JBEI Citizen” for two consecutive years for “fostering a positive working environment through helping colleagues.”

    “Perhaps Ee-Been’s most lasting legacy at JBEI is all the people she has helped along the way,” Beller said. “She’s been very generous with her time, including ongoing involvement in the iCLEM summer internship program for economically disadvantaged high-school students.”

    See the full article here .

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