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Genetic Modifications in Escherichia Coli and Pseudomonas Putida

Background: Hydrothermal liquefaction (HTL) is a technology used to convert algae biomass into “biocrude” oil a potential drop-in substance for petroleum. HTL eliminates land usage and provides a more complete biomass conversion than other methods. HTL produces an aqueous algae co-product (AqAl) which can contain residual carbon nitrogen and phosphorous that was initially present in the algae biomass. There is great interest in the development of methods to use these products for algae growth operations and thus recycle nutrients and enhance financial and material sustainability. Pseudomonas putida (P. putida) and Escherichia coli (E. coli) bacteria biomass mixtures can produce biocrude of a similar yield and quality as algae and an intermediate microbial growth step in the algae HTL process in which the organisms use the AqAl compounds as carbon and energy sources can improve the land footprint as well as reduce overall cost per volume of biocrude. However P. putida and E. coli can experience growth reduction in the presence of AqAl and consume only small fractions of the organic carbon present. Thus there is a need to improve the microbial growth rate and yield of P. putida and E. coli under these growth conditions for biocrude production. Technology Description: The developed technology is single nucleotide polymorphism (SNP) mutations which are small nucleotide insertions/deletions in P. putida and E. coli bacteria that are responsible for improved growth performance in AqAl media when compared to natural strains. Custom-designed bacterial strains containing one or a combination of these mutations could be used to increase the efficiency of a consolidated bio-mass-fed hydrothermal liquefaction process. Engineered P. putida and E. coli bacteria mutant strains outperformed their “un-evolved” parent by having a shorter lag time before growth a faster growth rate and/or an increased maximum cell density. Single SNP mutations in ilv PtsP and glpK genes which could respectively enhance metabolism improve tolerance to the carbon/nitrogen environment and interfere with the inactivation vital enzymes by nitrogen-containing compounds in AqAl were also developed for E. coli bacteria. These strains also showed improved growth characteristics when compared to their parent strain and demonstrate that each mutation found in the evolved strains could independently have a significant impact on AqAl utilization and tolerance. Applications: 1) Rapid and maximized growth of P. putida and E. coli - o Commercial o Research and development o Education o Biodefense o Medical 2) High yield production of “biocrude” oil - o Automotive o Commercial o Industrial o Manufacturing o Aviation o Aerospace

Benefits:

1) Reduced biocrude oil production costs 2) Incased hydrothermal liquefaction efficiency 3) Decreased hydrothermal liquefaction waste products 4) Lower land area use

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