Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

This report is available at no cost from the National Renewable Energy Laboratory (NREL) at www.nrel.gov/publications. U.S. Department of Energy (DOE) reports produced after 1991 and a growing number of pre-1991 documents are available free via www.OSTI.gov.NREL prints on paper that contains recycled content. ErrataThis report, originally published in August 2018, has been revised in September 2021 to correct life cycle assessment calculations applied to the biomass conversion via combined algae processing pathway analyses. The amended calculations and associated results were used to update Figures 22-28 and Tables 14 and A-1-A-4 in the Appendix. The changes to the results were not significant enough to impact any of the overall conclusions or trends outlined in the report, and did not impact resource assessment or techno-economic analysis metrics, nor life cycle assessment metrics for the hydrothermal liquefaction conversion pathway.iii This report is available at no cost from the National Renewable Energy Laboratory (NREL) at www.nrel.gov/publications. NomenclatureAD anaerobic digestion AFDW ash-free dry weight ANL Argonne National Laboratory BAT Biomass Assessment Tool BETO Bioenergy Technologies Office BGY billion gallons per year BGGE/yr billion gallons gasoline equivalent per year BT16 2016 Billion-Ton Report CAP combined algae processing CC carbon capture CHP combined heat and power CONUS conterminous United States DAP diammonium phosphate DOE U.S. Department of Energy FAME fatty acid methyl ester FFA free fatty acid FY fiscal year GAI Global Algae Innovations GHG greenhouse gas GREET Greenhouse Gases, Regulated Emissions, and Energy Use in Transportation HCSD high-carbohydrate Scenedesmus

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Harvesting corn stover for biofuel production may decrease soil organic carbon (SOC) and increase greenhouse gas (GHG) emissions. Adding additional organic matter into soil or reducing tillage intensity, however, could potentially offset this SOC loss. Here, using SOC and life cycle analysis (LCA) models, we evaluated the impacts of land management change (LMC), that is, stover removal, organic matter addition, and tillage on spatially explicit SOC level and biofuels' overall life cycle GHG emissions in US corn-soybean production systems. Results indicate that under conventional tillage (CT), 30% stover removal (dry weight) may reduce baseline SOC by 0.04 t C ha À1 yr À1 over a 30-year simulation period. Growing a cover crop during the fallow season or applying manure, on the other hand, could add to SOC and further reduce biofuels' life cycle GHG emissions. With 30% stover removal in a CT system, cover crop and manure application can increase SOC at the national level by about 0.06 and 0.02 t C ha À1 yr À1, respectively, compared to baseline cases without such measures. With contributions from this SOC increase, the life cycle GHG emissions for stover ethanol are more than 80% lower than those of gasoline, exceeding the US Renewable Fuel Standard mandate of 60% emissions reduction in cellulosic biofuels. Reducing tillage intensity while removing stover could also limit SOC loss or lead to SOC gain, which would lower stover ethanol life cycle GHG emissions to near or under the mandated 60% reduction. Without these organic matter inputs or reduced tillage intensity, however, the emissions will not meet this mandate. More efforts are still required to further identify key practical LMCs, improve SOC modeling, and accounting for LMCs in biofuel LCAs that incorporate stover removal.

Biofuels from microalgae are currently the subject of many research projects to determine their feasibility as a replacement for fossil fuels. In order to be a successful candidate, there must be enough fertilizers available to support large scale production. Commercial fertilizers are available for biofuel production from the world fertilizer surplus, but due to nitrogen and phosphorus future production limitations, biofuels would ideally not use any of these resources to be a long term sustainable fuel. Nitrogen, phosphorus and potassium requirements were determined for two algal species, Chlorella and Nannochloropsis, to produce 19 billion liters per year (BLPY). At this scale, both algal species would use 32-49%, 32-49% and less than 1% of the world surplus values of nitrogen, phosphorus and potassium, respectively. Nutrient recycling options and alternative sources of nutrients were evaluated to determine their potential contribution of lowering the synthetic fertilizer requirement. Results show that all of the recycling scenarios reduce the nutrient requirements, but catalytic hydrothermal gasification has the largest reduction of 95% of the nitrogen and 90% of the phosphorus. Contributions from all alternative sources can also provide only 5% or less of the required nitrogen when produced in the gulf region. For phosphorus in the same region, poultry concentrated animal feeding operations can provide up to 28% of the requirement of Chlorella. To find the least amount of nitrogen that may be used, catalytic hydrothermal gasification was combined with all of the alternative nutrients available in the gulf region. The maximum amount of biofuels that could be produced in this location without using any synthetic fertilizers is 50±20 BLPY from Chlorella and 45±19 BLPY from Nannochloropsis. This study shows that the nutrient requirement for biofuel production from microalgae will not be a limitation if recycling methods within the process chain and alternative sources of nutrients are utilized.

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