To the (Climate) Rescue: From First Blip in Chromatogram to Deployment at Industrial Pilot Scale

Industrial waste gas can serve as feedstock to manufacture fuels and chemicals through gas fermentation. Our work describes a collaborative and multidisciplinary team effort to engineer gas-utilizing microbe to produce acetone and isopropanol at industrial pilot scale in a carbon-negative fashion.
To the (Climate) Rescue: From First Blip in Chromatogram to Deployment at Industrial Pilot Scale

Back in 2008, in an underground laboratory in Auckland, New Zealand, an unconventional type of fermentation was brewing. Instead of utilizing sugars as carbon and energy source, a group of scientists and engineers was training microbes known as acetogens to gobble up waste gas (containing CO2, CO and H2) from a local steel mill, and spit out fuels and chemicals. Fresh out of university, I was amongst the first 20 employees of LanzaTech and was tasked with characterizing these acetogens at the molecular level and developing genetic tools to make novel chemicals. The entire field of gas fermentation was in its infancy and literature on successful genetic engineering of acetogens was non-existent. Dr. Michael Köpke and Dr. Ching Leang are early pioneers in this field and both joined LanzaTech to propel this field to new heights.

Fungmin Eric Liew (left) and Robert Nogle (middle) working in an anaerobic chamber; Tanus Abdalla (right) attending to a CSTR at LanzaTech Skokie R&D facility.

One of the first tasks that I was given when I joined LanzaTech was to screen an industrial clostridial collection of ABE (­Acetone-Butanol-Ethanol) strains reaching back to the 1940s assembled by Prof. David Jones (University of Otago). Little did I know that this microbial collection would one day be genome-sequenced (by the DOE Joint Genome Institute) and form the basis of a multidisciplinary collaboration between Northwestern University (cell-free prototyping and kinetic modelling), Oak Ridge National Laboratory (multi-omics) and LanzaTech to develop a gas-to-acetone/isopropanol process.

LanzaTech scientists guarding the clostridial collection (also dubbed the ‘Davy Jones locker’). From left to right: Michael Köpke, Sarah Ye, Rasmus Jensen, and Steven Brown. Inset: Original soil spore stocks from the ‘Davy Jones locker’ in different format.

From its early conception, the founders of LanzaTech recognized the potential of gas fermentation to go “beyond ethanol” through synthetic biology approaches. In 2011, Dr. Michael Köpke, Dr. Sean Simpson, and I filed our first patent applications demonstrating the successful genetic engineering of acetogens to produce non-native molecules, such as n-butanol and acetone by introducing ABE genes. The acetone generated only a small ‘blip’ in the chromatogram at the time, and even though there was enormous excitement it was clear that more extensive R&D effort was required for commercialization.

Published in this month’s Nature Biotechnology, our paper Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale” describes the use of genes mined from the David Jones collection to construct an acetone combinatorial pathway library. The genome of acetogen used in this study, Clostridium autoethanogenum, encodes a secondary alcohol dehydrogenase which further reduces acetone to isopropanol. Both molecules are industrial solvents as well as platform chemicals for the production of materials such as acrylic glass and polypropylene. Once the optimized gene combinations were determined, Robert Nogle and Dr. Ching Leang led the effort in generating integrated strains for improved strain performance and stability that are prerequisites to a scale-up process. Similar to myself, Robert joined LanzaTech fresh out of university, in 2014. Robert’s been a quick learner of the unusual anaerobic techniques and quickly made his mark and was quick to implement solutions for identified bottleneck and construct next iterations of strains. To our knowledge, the final versions of the acetone and IPA producing strains are the most manipulated acetogens to date.

During the characterization of early iterations of acetone strains, it was discovered that off-pathway reactions led to the formation of unwanted side products due to interaction with native metabolism. To guide the “finding the needle in the haystack” effort of identifying responsible genes for this byproduct formation, we worked with our partner Dr. Michael Jewett’s laboratory from Northwestern University and employed cell-free prototyping to uncover this at a speed that is not possible in vivo. Indeed, this helped reduce the time for synthetic biology design from many months/years to weeks. Omics measurements performed by our partners from ORNL and kinetic modelling provided unique insights that suggested the CoA transferase reaction as a limiting step for acetone production. The end result of incorporating all these multidisciplinary tools and techniques are optimized strains that are highly selective for acetone or isopropanol.

Tanus Abdalla led the effort in performing bench-top continuous gas fermentation in 2L continuously stirred tank reactor (CSTR) using acetone and isopropanol strains generated in this study. Tanus is a biochemist who first joined LanzaTech NZ as an intern in 2012 before moving to a full-time position. In addition to screening strains and running well controlled CSTR to generate data for the omics experiment, he also optimized the fermentation parameters to improve the production rates and selectivity. At the later stage of the project, he supported the fermentation scale-up effort by transferring the learnings from the CSTR to the pilot scale. After hundreds of fermentation runs via collective team effort, the engineered strains demonstrated production at rates of up to ~3 g/L/h and ~90% selectivity using synthesis gas as feedstock. Eric, Robert and Tanus are now senior researchers and project leaders within LanzaTech. Besides their role as scientist, Tanus and Robert led the effort to form an initiative called “Blend” to empower inclusion, diversity and equity at LanzaTech’s workplace.

Since 2014, LanzaTech is headquartered in Chicago, IL, and currently operates two full-scale commercial plants converting industrial off-gas to ethanol with a by-product of high protein biomass (for animal feed), with 7 plants planned to come online across the globe by year 2022, utilizing a variety of feedstocks including municipal solid waste, agricultural waste, and carbon emissions from industries. In addition to being a drop-in fuel, ethanol has been purified and upgraded into a range of CarbonSmartTM products that are accessible to today’s consumers: fragrance, household cleaning products, sustainable packing and fashion. Furthermore, ethanol has been chemically upgraded to paraffins and isoparaffins for use as sustainable aviation fuel and used in two transoceanic flights in 2018 and 2019. Scaling up bioprocesses is challenging and it’s been a privilege to see microbes developed in the lab being deployed at half million-liter tanks around the world and we are excited to see acetone and isopropanol strains deployed next in these plants.

Life cycle analysis (LCA) confirmed a negative carbon footprint for the products. To limit global warming to less than 1.5 oC relative to pre-industrial levels, a multitude of renewable technologies, as well as change in human behavior, is required. We believe gas fermentation represents one such technology that can close the global carbon loop. The U.S. Department of Energy through it's Biosystems Design Program led by Dr. Pablo Rabinowicz and MegaBIO program led by Dr. David Babson and Dr. Jay Fitzgerald provided important support for the project. This paper highlights the importance of assembling a collaborative and multidisciplinary team to accelerate and achieve that target, one carbon molecule at a time.

Left: cBioFab Team featuring scientists from Northwestern University, LanzaTech, and Oak Ridge National Laboratory; Right: Synthetic Biology Team at LanzaTech.

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