This study explored transient inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of MutS deletion derivatives of 12A and ATCC 334 were constructed and subjected to a 100-day adaptive evolution process to increase lactic acid resistance at low pH. Wild-type parental strains were also subjected to this treatment. At the end of the process, the Δ lesion was repaired in representative 12A and ATCC 334 Δ mutant isolates. Growth studies in broth at pH 4.0 (titrated with lactic acid) showed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. However, the adapted Δ derivative mutants showed the greatest increases in growth and lactic acid production. Further characterization of the 12A-adapted Δ derivative revealed that it had a significantly smaller cell volume, a rougher cell surface, and significantly better survival at pH 2.5 than parental 12A. Genome sequence analysis confirmed that transient inactivation decreased DNA replication fidelity in both strains, and it identified genetic changes that might contribute to the lactic acid-resistant phenotypes of adapted cells. Targeted inactivation of three genes that had acquired nonsense mutations in the adapted 12A Δ mutant derivative showed that NADH dehydrogenase (), phosphate transport ATP-binding protein PstB (), and two-component signal transduction system (TCS) quorum-sensing histidine protein kinase () genes act in combination to increase lactic acid resistance in 12A. Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes, including acid resistance. We developed a method to increase the adaptability of 12A and ATCC 334 through transient inactivation of the DNA mismatch repair enzyme MutS. Here, we show this method was effective in increasing the resistance of to lactic acid at low pH. Additionally, we identified three genes that contribute to increased acid resistance in 12A. These results provide valuable insight on methods to enhance an organism's fitness to complex phenotypes through adaptive evolution and targeted gene inactivation.

De-oiled algal biomass (algal cake) generated as waste byproduct during algal biodiesel production is a promising fermentable substrate for co-production of value-added chemicals in biorefinery systems. We explored the ability of Lactobacillus casei 12A to ferment algal cake for co-production of lactic acid. Carbohydrate and amino acid availability were determined to be limiting nutritional requirements for growth and lactic acid production by L. casei. These nutritional requirements were effectively addressed through enzymatic hydrolysis of the algal cake material using α-amylase, cellulase (endo-1,4-β-D-glucanase), and pepsin. Results confirm fermentation of algal cake for production of value-added chemicals is a promising avenue for increasing the overall cost competiveness of the algal biodiesel production process.

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