Publishing in respected peer-reviewed scientific journals gives researchers the opportunity to showcase their work to others at the forefront of science and affirms the quality and significance of their accomplishment. This year, Science, Nature and Proceedings of the National Academy of Sciences featured research conducted at EMSL, the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility at Pacific Northwest National Laboratory.
EMSL staff and visiting scientists use highly specialized instruments and high performance computing, or HPC, to investigate critical research questions. “We offer unique expertise and capabilities together under the same roof to produce significant scientific discoveries,” says EMSL Director Liyuan Liang.
This year, readers of these top-tier journals have learned about research done at EMSL that led to developing cells that produce more lipids for use as biofuels; identifying fungal genes that promote more efficient digestion of cellulose; elucidating enzyme pathways in methane synthesis; and explaining how microbes affect fracking.
Unique, Just Like Every Other Cell
continuous laminar flow. (Image from Andreas Vasdekis,
University of Idaho)
Microbial strains with genes modified to produce a large amount of lipids offer a promising route for efficiently producing biodiesel. These fatty, energy-rich molecules are a key ingredient in this fossil fuel alternative. Engineering a lipid-rich microbe follows the same principle as modifying any organism to produce more of a desired trait, such as an apple tree that produces fleshier fruit.
Experimental modification of microbes' DNA to produce more lipids often does not reach the yields predicted by theory. That creates an obstacle impeding the goal of finding efficiencies in the manufacture of biofuels.
With Gregory Stephanopoulos and colleagues at MIT, EMSL staff scientist Andreas Vasdekis, now at the University of Idaho, explained in a Scientific Reports paper why theory and experiments disagree. By observing one cell at a time, they discovered no two cells are the same in producing lipids.
Researchers typically work with billions of cells examined in bulk. In contrast, the EMSL team observed multiple isolated cells separately in extremely small, stable volumes of fluids. They used high-resolution microscopes to image the cells and identify chemical compounds in their environment. They found lipid production fluctuated sporadically in multiple strains of both high-yield and low-yield organisms.
“It's like taking a room full of people and measuring average fat content of each person instead of assuming everyone is exactly the same," says Vasdekis.
It's commonly assumed all cells in a culture experience the same environment, but they don't. The study isolated individual cells and showed differences in the activity of key metabolic genes that contribute to lipid production, with environment playing a role.
“The single cell gives us insight to what regulates the metabolism of lipid production,” adds Vasdekis. This insight is a step forward to improve engineered strains for developing lipids as an efficient method of producing a fossil fuel alternative.
Clever, Crafty Fungi
To create biofuels from plant-derived precursors such as woody cellulose, manufacturers first need to break down these organic materials to extract energy-containing components, often by harnessing digestive processes of living organisms.
Brown rot fungi are renowned for their ability to deconstruct cellulose using remarkably few enzymes, but how they control their unique digestion has puzzled scientists. One hypothesis is fungi produce highly reactive chemical agents to pre-treat the tough cellulose, making it easier to digest with fewer enzymes.
Mycologist Jonathan Schilling at the University of Minnesota collaborated with EMSL's Galya Orr and Dehong Hu to identify brown rot fungi's oxidative agent mechanism. These chemicals strip electrons from a material to degrade it without damaging the fungi or the secreted enzymes. Their PNAS paper explains how expression of one set of genes controls this oxidative step, before another set of genes turns on the traditional digestive enzymes.
Schilling and EMSL staff observed fungi growing along a piece of wood. They found fungi pre-digested tough lignocellulose with oxidative chemicals in wood samples taken early on. In later specimens, they saw genes and enzymes typical of decomposer fungi deployed to complete digestion.
Using microscopy techniques and fluorescent labels to identify specific genes, they could tell some genes turned on at the 'front' of the organism but were not turned on further behind. Many of these genes play a role in the reactions that begin to break down woody materials in advance of the enzymes.
“The genes involved in the oxidative step are ... a pretreatment of the biomass," says Schilling. "They're happening in the same piece of wood as traditional enzymes, but they're segregated by space.”
This unusual step offers a shortcut for producing an enzyme cocktail with fewer ingredients. As Schilling notes, “It's a clever, crafty upgrade from a gutless organism: they're producing oxidative agents we humans wouldn't want in our stomachs, and using them selectively.”
Schilling’s next focus includes collaborating with bioinformatics scientists at EMSL to discover how the genes are actually regulated. The ability to splice those key genes into other bacteria and fungi may offer a path for increased efficiencies in breaking down cellulose, which is of interest to the biofuel industry.
Elucidating Enzyme Pathways in Methane Synthesis
Another hydrocarbon fuel, methane, is gaining importance due to its high energy density – but it is also a powerful greenhouse gas. Scientists struggle to comprehend the mechanism single-celled organisms use to produce 90 percent of Earth's atmospheric methane. They want to understand natural methane generation to develop better catalysts for manufacturing it by mimicking the enzymes used by methane-producing microorganisms.
Stephen Ragsdale at the University of Michigan with PNNL scientists Dayle Smith and Simone Raugei identified the intermediate step in the enzymatic formation of methane and published their findings in Science.
Using spectroscopic tools at EMSL, they identified the intermediate compound that leads to synthesis of the enzyme used by microbes to catalyze methane formation. Then, using HPC resources at EMSL and at Lawrence Berkeley National Laboratory’s National Energy Research Scientific Computing center, another DOE Office of Science User Facility, the team verified this intermediate compound is favored over other potential candidates.
“This study identified an important mechanism nobody understood before," says Liang. "When you understand the mechanism, you can try to manipulate it and produce biomimetic catalysts.”
This discovery may result in better catalysts for conversion of methane into fuels as well as biofuels production. The findings could also lead to the development of better strategies to inhibit microbial production of the potent greenhouse gas.
Microbial Life Thrives on Fracking
cells that thrive in fracking wells. (Image from Mike Wilkins,
courtesy of The Ohio State University)
Hydrocarbons remain a major source of the world's energy despite efforts to diversify energy sources. Hydraulic fracturing, or fracking, of deep shale formations is a method for recovering natural gas.
One challenge of this method is how best to keep microbes from colonizing downhole reservoirs where they corrode and clog extraction systems. Operators introduce chemicals to kill microorganisms, but it's unclear how successfully they keep a reservoir microbe-free.
Scientists Kelly Wrighton and Mike Wilkins from The Ohio State University collaborated with EMSL biochemist David Hoyt to investigate how the microbial populations of two Appalachian shale formations fared during fracking.
Quite well, it appears, as reported in Nature Microbiology.
Wilkins summarized the complexity of the problem, “Industry spends a lot of time and money trying to kill microorganisms. But this is a huge ecosystem: it's one thing to pasteurize a bottle of milk; it's another thing to pasteurize a subsurface reservoir.”
Fluid samples from the wells showed microbial populations inadvertently introduced during the fracking process colonize the deep subsurface and can thrive downhole. The researchers reconstructed the genomes of these bacteria to reveal their adaptations to extreme chemical conditions using a combination of nuclear magnetic resonance probes at EMSL and DNA sequencing technologies at the DOE Joint Genome Institute, a DOE Office of Science User Facility at Lawrence Berkeley National Laboratory.
“We learned microbial life is tougher than people want to admit,” adds Hoyt.
The team developed a synthetic ecosystem. They will use it to investigate specific biochemical pathways microorganisms use to adapt to the extreme conditions produced by fracking. Industry and basic energy research alike await the results.
User Support for User Facilities
EMSL staff expertise and capabilities provide investigators with opportunities to optimize their research and hone techniques for handling diverse samples from many disciplines under one roof. “Having both the NMR and spectroscopic tools ... let us answer questions at the interface of geochemistry and microbiology,” says Wilkins.
“EMSL had me collaborating with people who not only had their hands on the microscopes, but also had a background in biochemistry,” says Schilling.
“The message is -- our research is world-class,” concludes Liang.
Rachel Berkowitz is a freelance writer.
Funding: DOE’s Office of Science supported all four studies. The microbial lipid production study in Scientific Reports also received support from the National Institute of General Medical Sciences of the National Institutes of Health, and a Linus Pauling Fellowship from PNNL. The brown rot fungi study received support from the Advanced Research Project Agency – Energy.
EMSL, the Environmental Molecular Sciences Laboratory, is a DOE Office of Science User Facility located at Pacific Northwest National Laboratory in Richland, Washington. EMSL offers an open, collaborative environment for scientific discovery to researchers around the world, and its integrated computational and experimental resources enable researchers to realize important scientific insights and create new technologies.