Industrial revolutions are rare, but when they come, they change everything. We are at the start of just such a time now. The convergence of synthetic biology, robotics, and biomanufacturing is ushering in a new bioeconomy that is based on biotechnologies rather than petrochemical production.
The Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is expanding its biomanufacturing capabilities to be the tip of the spear in this effort – and in the process, providing a big job boost by helping to establish a bioeconomy in the U.S. and Maryland.
What is Biomanufacturing?
“Currently, the critical chemicals the nation needs both for national security and to sustain the 21st Century economy are manufactured in costly petrochemical facilities,” said Dr. Peter Emanuel, the Center’s senior research scientist for bioengineering and overall leader of the DEVCOM CBC biomanufacturing initiative. “Many crucial chemicals are either manufactured by a single source domestically, or worse yet, inside foreign nations that may not always be willing to supply us. Biomanufacturing is a manufacturing revolution that can make the United States self-sufficient and far more sophisticated in its chemical manufacturing.”
Biomanufacturing enables the creation of high-performance, high value materials by controlling the genetic makeup of the organisms that make them. Much like a microbrewery that produces craft beers, biomanufacturing uses bacteria in vats to ferment new materials. However, biomanufacturing is not entirely a new concept – the use of microorganisms to produce military-relevant products dates back to 1916, when bacteria were first used to produce acetone that was critical for the manufacture of explosive propellants. But today, genetic engineering tools such as CRISPR/Cas9 and the interdisciplinary field of synthetic biology have given us an unprecedented degree of control over microbial metabolism. Using those engineering tools, we can specify DNA sequences in a microbe to direct the production of new materials, each with highly specialized and valuable properties.
“The critical chemicals that domestic, low-cost biomanufacturing can produce include energy-dense propellants and explosives, reactive coatings and textiles, optical and sensor materials that can bend light, and new therapeutics such as antimicrobials and vaccines,” said Dr. Henry Gibbons, a DEVCOM CBC microbiologist and program manager of the Center’s current biomanufacturing expansion effort.
How is it Done?
“At the Chemical Biological Center, it starts with a microbial cell, an organism so small it must be viewed under a microscope that grows quickly and readily accepts changes to its genetic template, the DNA that serves as the molecular instructions to control its activities. This lets researchers quickly modify cells to unlock access to a wide variety of desired chemical products,” said Dr. Anna Crumbley, a National Research Council postdoctoral fellow recently hired by the Center for her expertise in engineering bacteria.
Center scientists will use strains that have been modified using cutting edge molecular engineering technology to customize the structure of a microbe’s DNA. Once inside the cell, the DNA is read and acted on to change the cell’s behavior – just like opening a new app on your smartphone. The engineered DNA allows scientists to take control of the cells’ manufacturing processes so that the cells are now manufacturing a high-value material.
Microorganisms can be directed to react with and neutralize a chemical agent, or transform sugar-based feed chemicals to form other molecules, such as generating a propellant, coating sensor material, or a new therapeutic such as a vaccine.
“Biomanufacturing allows for agility and diversity that cannot be achieved in traditional petrochemical production,” said Dr. Jared DeCoste, a research chemist who has a key role in coordinating the Center’s work with other scientists and laboratories that are currently developing DNA sequences to make new materials. “For the DoD, there is a need to be producing small batch specialty chemicals, which means that we may be producing one chemical or material today, but need to shift to something else quickly. Traditional petrochemical production does not allow for this, but with biomanufacturing you can turn over your equipment quickly and introduce a new genetically modified organism to produce your desired product.”
While in theory almost any microorganism can be used for biomanufacturing, the living cells of choice are workhorse laboratory strains of brewer’s yeast and Escherichia coli because they are very simple and safe for scientists to work with at scale.
What Biomanufacturing Looks Like
Walking into the Center’s biomanufacturing facility, what one notices right away is lots of large, shiny steel vats all side by side, just like in a microbrewery. But those 1,000-liter vats are only one part of a very precise manufacturing sequence. It all begins in a small petri dish. The carefully engineered microbe is placed on the dish and grown over a course of days until there is enough to fill a flask containing a broth of sugars that serve as food for the microbes. That flask is then placed on an auto-shaker to ensure even growth.
The microbe population in the flask increases until there are enough of them to put in the first fermentation vat. It is only 20 liters. It contains a larger volume of the broth plus oxygen which is steadily pumped in to further stimulate microbial reproduction. This process can take anywhere from hours to days depending on what kind of microbe is being grown.
The microbe stew continues to thicken until it is dense enough to pipe over to a large fermentation vat. There, the microbes continue to grow in the oxygenated broth until the time is right to extract the microbes. The end product needed to create a new material may be the microbe itself. But it can also be a part within the microbe such as a lipid or a protein. Whatever the active ingredient is, it needs to be extracted from the broth.
Gibbons explains the first extraction step by saying, “think of a really big salad spinner.” He is referring to a centrifuge, to which the broth containing the mature microbes is pumped. A bucket inside of it spins at high speed until the microbes are pressed against the outer wall of the centrifuge, and are ready to be removed.
But that is not the only way to perform extraction. A different device allows Gibbons and his team to remove these highly valuable microbes in a process that looks like a giant French press used for making coffee. Whichever method is chosen, the end result is pure broth inside the vat and an extraction of pure microbes. This completes what Gibbons refers to as “the front end of the process” which is followed by “downstream processing.”
Downstream processing can take any one of several different forms, depending on where the desired product is. If the microbe itself is the product, the microbial paste can be freeze dried, much like coffee, and later added to a solution so that it revives and begins to perform its material-making function. It can be baked at high temperature and turned into a powder that then can be turned into pellets to be used as a filtration material in gas masks or turned into a sprayable decontamination powder for surfaces, or it can be woven into long fibers to form textiles that will neutralize chemical agents on contact. Or the microbes can be rendered into a dry spray much like powdered milk or baby formula that is made functional by placing it in water and heated. If the desired product is inside the microbe, they can be smashed open and turned into a liquid. The product can be extracted from liquidified microbes or from the broth itself using various filters or solvents.
In order to accommodate this range of of downstream processing, the Center will be equipping a processing center within the biomanufacturing facility. Gibbons anticpates that within two years the Center will be able to perform many different types of downstream processing, including distillation with distillation columns as high as 35 feet. It will be modular and reconfigured as needed to best perform the exact type of processing required for the materials being produced.
Biomanufacturing Economics
So how expensive is it to manufacture these microbes? According to Gibbons, it depends on several different factors. How expensive is the nutritional broth being used? How much energy will be needed to heat the broth and boil down the water in the vats? How much product will be available for use at the end of the processing? Can the left over broth be recycled, or does it have to be disposed of as a hazardous material?
Gibbons and his team are working on refining the answers to these questions by combining their production work with what they call “techno-economic analysis.” What guides the analysis is a comparison of the costs of biomanufacturing to the cost of conventional industrial production, typically in petrochemical plants. These calculations have to include the larger societal cost of pollution generated by petrochemical processes.
“We use this analysis to be able to tell potential customers in the defense and commercial sectors up front, can we do what you want done economically,” said Gibbons. “That gives them a clear basis for making a go/no-go decision on going forward with production.”
Why Biomanufacturing is Important
The economic impacts of COVID-19 will last long after vaccines bring a new normal and begin the long road to recovery for the U.S. economy. One unexpected effect of the pandemic is that it exposed a weakness in U.S. manufacturing that has been building for years. U.S. industry’s distribution system and supply chains were vulnerable before COVID-19, but pandemic-related disruptions to global supply chains fully exposed that already existing problem. U.S. manufacturers have relied too heavily on foreign materials for production, and the steady off-shoring of critical industries over a course of decades has reduced the nation’s direct control of vital defense-related manufacturing. Therefore, for national security reasons the U.S. must invest in an economic and technological acceleration program that focuses on growing America’s ability to supply its industrial sector with domestically produced biomanufactured products.
Chemical Biological Center scientists’ skills combined with its 100-plus year history as the DoD’s brain trust for chemical and biological research and innovation makes it uniquely qualified to take on this role. Center scientists see an opportunity to directly benefit the warfighter, too. In future conflicts, the equipment they need that comes from newly invented high-value chemicals may not have to be manufactured in the U.S. and sent to the battlefield through a long supply chain. Instead, in the future, it may be possible to place the bioproduction facility needed for production near the front. Then supply is just a matter of having someone bring a cooler full of test tubes containing samples of customized microbes, which produce the needed material, right there near the front line. The material goes into production in the vats right there on an on-demand, rapid turnaround basis.
What Makes The Chemical Biological Center Unique?
Academic and other Department of Defense research laboratories are synthesizing microbes with extraordinary properties, but only at the gram-sized scale. Production at this bench scale is only the first tentative step toward being able to produce commercial quantities of a product. The Center provides an intermediate or pilot step in which scientists solve the inevitable technical challenges that come with scaling-up production to commercial quantities. Sometimes the performance characteristics of the engineered microbes change with scale.
Making 1,000-liter batches just isn’t the same as what fundamental science researchers do using a flask on a shaker in their own laboratory,” said Crumbley. “Growing the same microbes in a large tank means having to control for physical stresses, such as the shearing of the cells as they are mixed in the large vat of liquid. Also, when you have billions of microbes, they generate their own heat as they grow. That has to be included in our decision of how much mechanical mixing we are going to do. Those are just two of many considerations we have to take into account.”
“A facility like this is only as good as the people who operate it, and we have the best.”
Dr. Nicole Rosenzweig
The Center’s biomanufacturing team also has to do a lot of monitoring in order to stay on top of all these factors. Sensors inside the vats track the temperature of the liquid, the mixing speed, the diffusion of oxygen, and the pH and many other things. All of this data appear on computer screens next to the vats, which they constantly scan. They make adjustments as needed to optimize microbe growth and avoid problems such as oxygen-deprived microbes, over-overheated microbes, or over-agitated microbes. Any of these problems can cause the microbes to lose the integrity of their carefully designed chemical production sequence.
What makes the members of this team so unique is that they have that rare combination of chemical engineering and molecular biology knowledge needed to use all that incoming data to finesse the fermentation process. “We have taken great care to hire just the right people,” said Dr. Nicole Rosenzweig, the Biosciences Division Chief who assembled the team. “A facility like this is only as good as the people who operate it, and we have the best.”
In effect, the Center functions as a midwife between producing bench-scale quantities of new chemicals and producing large-scale production quantities. To help advance this emerging technology, the Defense Advanced Projects Agency (DARPA) funded a Center demonstration project in 2020 to produce BioCNFs, which is short for biologically-templated nanofiber. It is a phage, a virus that infects bacteria, and it can be used to neutralize chemical warfare agents either as pellets within a gas mask filter or as a sprayable decontamination powder. The Center has been producing BioCNFs for several years, but with DARPA’s investment, the Center can go from grams up to kilograms.
Also with DARPA funding, the Center will be working with Pennsylvania State University to create a self-healing fabric using protein taken from a squid tooth. This is but one example of the kind of previously unattainable chemicals which will be a boon to national defense and American industry as a whole. The number of laboratories that can fill this specialized role is very small. The Center joins only two other U.S.-based facilities that have a similar capability, the University of California at Berkeley and at Michigan State University. Both these facilities have far more demand than they can meet from companies hoping to commercialize their own microbial products.
“Having proved the concept, the next step for the Center is to become the go-to place for the DoD to be able to develop highly advanced coatings, adhesives and polymers, novel fuels, and liquid crystals for a wide range of defense needs,” Emanuel said.
DARPA agrees. Having established what they call the Living Foundries Program, they see the Center’s biomanufacturing initiative as part of a larger effort to help create a 21st Century bio-economy that will develop a wide range of high value chemicals for defense and civilian industrial uses. DARPA sees the Center as “a reliable resource to routinely and robustly scale and deliver molecules and materials for advanced development,” according to an August 2019 letter from DARPA to the Deputy Assistant Secretary of Defense for industrial policy supporting funding for the Center’s pilot plant.
The Center is now starting a new phase of biomanufacturing that will solidify its place as the go-to facility for biomanufacturing scale-up. The Center is slated to receive $24 million over 5 years from the U.S. Army to renovate and expand its current facility.
This is necessary because of the limits to its current production capabilities. Many chemicals generated through biomanufacturing in a watery microbial growth medium are very difficult to remove from the fermentation broth, or are insoluble in water. They have to be extracted using chemical rather than mechanical methods. This will broaden the range of high value chemicals that the Center can manufacture.
This requires larger equipment and a more elaborate set of safety protocols than aqueous-only manufacturing. What will remain the same is that there is no need for high pressures and temperatures to drive chemical reactions as with conventional petrochemical manufacturing facilities. Fermentation operates at ambient pressure and temperatures, making it far safer and eco-friendly. In addition, the same set of vats can be used to manufacture a given chemical, then after being cleaned out, manufacture an entirely different chemical.
Biomanufacturing Missile Fuel
As both a larger proof of concept and to meet an immediate defense need, the initiative’s first project will be to biomanufacture 1,2,4-butanetriol (BT), a critical precursor to butanetriol trinitrate (BTTN), a fuel used in Hellfire missiles. Center scientists will use genetically modified microbes to produce BT in its production facility at APG and will partner with other DoD laboratories to produce the final BTTN product. DoD currently relies on a single domestic supplier of BT and needs to diversify production.
Establishing an alternative domestic source for this fuel is important because the Hellfire is the U.S. military’s weapon of choice for precision strikes on high-value targets. It travels at 1,000 miles an hour and has a range of nearly seven miles. It can be launched from attack aircraft or drones and has been instrumental in eliminating a number of high-profile terrorists in the Middle East.
BTTN is not only used in the Hellfire, but in virtually all single-stage missiles used by the United States because it is less volatile, less sensitive to shock, and more thermally stable than the alternative, nitroglycerine. The Office of Naval Research began funding an effort to biomanufacture the fuel in 2004 at Michigan State University. The U.S. military has now turned to the Center to scale up the process.
Next Steps
The Center’s biomanufacturing facility will be fully constructed and ready to run at full capacity within two years. At that point, the Center can market itself throughout DoD as the go-to place for enhancing and manufacturing high value chemicals of military value. Furthermore, the Center will have demonstrated that it is a central partner in the overall defense goal of establishing manufacturing independence from foreign suppliers. This places the Center in a key role for DoD’s long-range goal of developing America’s bioindustrial base. Pentagon planners call this the Bioindustrial Manufacturing and Design Ecosystem, or BioMADE.
“The sky is the limit for the kind of advanced manufacturing the Center will be able to support,” said Emanuel. Among the possibilities are:
Living textiles for environmentally interactive uniforms
Bending light for bio-camouflage on warfighters, vehicles, and aircraft
Decontaminating sunscreens that last a lifetime with a single application
Generating high octane rocket fuels without a conventional refinery
Heat resistant protective surfaces for hypersonic missiles
Unjammable bioelectronic computing components
The ongoing bioindustrial revolution is characterized by the blurring of the divisions between the physical, digital, and biological domains. It will rapidly transform global relationships and disrupt all prior industrial technology. China is likely to be America’s chief competitor in what may prove to be a superpower manufacturing technology race in which staying ahead will be a national security imperative. The DEVCOM Chemical Biological Center’s role is to provide the expertise and the production capability to ensure that the U.S. leads the technology race as biomanufacturing changes the world.
Anna Crumbley Brings New Biomanufacturing Expertise to the Chemical Biological Center
By Dr. Brian B. Feeney
Where does a newly minted doctoral graduate in metabolic engineering and industrial manufacturing go to make an impact on the world? In the case of Anna Crumbley, it was the U.S. Army Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC), to become a key member of the team that is developing a biomanufacturing capability that will help usher in a new economy based on biotechnologies rather than petrochemical production.
She joined the Center in August as a National Research Council (NRC) Research Associateship Program Fellow and is now a key member of the Center’s biomanufacturing initiative. She was recruited for her experience with engineering bacteria to produce higher value chemicals. Her efforts will be focused on scaling up strains of microbial organisms that other laboratories send to the Center as well as fine-tuning these microbes so that their intended functionality is enhanced.
“When I got to high school, the BP oil spill made a big impression on me and I wanted to be part of a better, greener way society could manufacture the materials it needs,”
Dr. Anna Crumbley
It seems that she was always meant for science. Her parents and both her grandfathers were engineers at NASA. They worked on sending Apollo rockets to the moon, launching the Space Station, and many other projects. The family trait showed itself in her early on.
“As far back as elementary school I would play with Steve Spangler Science Kit projects and make things like colored jelly crystals using food dye and a version of Silly Putty called flubber, made with Elmer’s glue and boric acid,” she said. In middle school, she spent summers as a participant in the DEVCOM Aviation and Missile Center’s Gains in the Education of Mathematics and Science, or GEMS, program enabling students to experience science and engineering in real-world Army laboratory settings.
“When I got to high school, the BP oil spill made a big impression on me and I wanted to be part of a better, greener way society could manufacture the materials it needs,” Crumbley said. She was fortunate enough to have a chemistry teacher at her high school in Madison, just outside Huntsville, Alabama, who got her excited about biology and all the things it could do for the world.
In 2011, she enrolled at the University of Alabama, graduating in 2015 with a degree in chemical and biological engineering. After spending several semesters as an undergraduate research assistant in a University of Alabama biotechnology laboratory, she wanted to learn more about biotechnology and enrolled in a Ph.D. program at Rice University in Houston, Texas, where she could learn from some of the best researchers in the nation in that highly specialized field.
“In my Ph.D. program I learned to think of a cell as a small chemical reactor, with the enzymes inside it as tiny catalysts. By changing the organism’s genetics – its DNA and enzymes – we can essentially change the organism’s digestion, and as a result get it to transform sugars and some nitrogen into all sorts of useful chemicals,” she said. “So at Rice I was combining the science of biology with the technical aspects of chemical engineering. I’ve long had a fascination with microbiology, but chemical engineering gave me a great application for it in the form of bioengineering.”
As she was getting ready to graduate from Rice University she remembered a conversation she had at a science convention three years earlier where she met an NRC Research Associateship Program Fellow working in a related field. This person told Crumbley that it was a good experience and that she should consider entering science through that path, too. So she went to the NRC website and quickly found a research opening under the program being offered by Dr. Peter Emanuel, DEVCOM CBC’s senior research scientist for bioengineering and overall leader of its biomanufacturing initiative.
She e-mailed him, and sensing the value of her skills, he responded very quickly. In a phone interview, he explained to her what the Center was endeavoring to do, and the prospect of helping to create entirely new materials using what she had learned in her studies held a strong appeal. Later, touring the 20,000-foot biomanufacturing facility at Aberdeen Proving Ground and meeting the other members of the team only increased her enthusiasm. “I had always worked at the bench scale level in graduate school, so to see a team of researchers making batches of more than 1,000 liters, about the size of a craft beer brewery tank, was very exciting. I also appreciated the excitement the members of the Center’s team had about what they were doing,” she said.
Several months into her job, she is as glad as ever to be a part of the initiative. “Being able to do this kind of work at this scale, and advance the research of other laboratories around the country by scaling up their discoveries, I couldn’t have hoped for more,” she said.
Emanuel is glad that she is at the Center, too. “I’m really excited for Annie to join this effort. She is a strategic hire in our long-term initiative to establish the Center as a trusted partner within the synthetic biology community, serving all branches of the armed forces,” he said. “And, what was an amazing bonus was her skill set aligns directly with some of the larger biomanufacturing projects that we have planned for the next several years. I’m excited that Annie is going to be able to play such a prominent role in making those a reality.”
Partnership Provides Opportunities in Biomanufacturing Training for Local Students
Harford Community College (HCC) is the first of what is expected to be several Maryland colleges and universities to enter into an educational partnership with the Chemical Biological Center. These partnerships are designed to prepare students to enter the region’s rapidly growing biotechnology workforce.
Dr. Eric L. Moore, director of the Chemical Biological Center, and HCC’s new president, Dr. Theresa Felder, signed an agreement on February 10 to lay the groundwork for a biomanufacturing cooperative educational program.
Under the agreement, the Center and the College will establish a cooperative hands-on training program to provide students with access to Army subject matter experts and state-of-the-art facilities in biomanufacturing, synthetic biology, biotechnology, and data analysis architecture. Students will have the opportunity to begin their industry careers through work-based apprenticeship learning experiences, and the Center will gain access to a talent pool with the professional, field-specific, and career-readiness skills.
The Center is close to establishing the same kind of partnership with Cecil College. The Center has also begun discussions with UMBC at the Universities at Shady Grove and Johns Hopkins University.
“The benefit of getting agreements with these two four-year colleges and universities is that we can help establish a pipeline from HCC and Cecil College’s two-year programs in bioproduction to a final two years in biotechnology leading to a bachelor of science degree,” said Dr. Jared DeCoste, a Center research chemist who has taken the lead in forming these partnerships. “In addition to providing guest lecturers who have an unrivaled expertise in this field, we will also provide hands-on apprenticeships during the summer and during the academic year, giving students a broader view of the biotechnology industry. Ultimately, some of these students may have an opportunity to work at the Center.”