Screening of spore forming d-lactic acid producing bacteria and its optimization
Authors:   , Prasirtsak Budsabathip 
Published: September 2023

In this study, isolation, screening, fermentation optimization, and determination of the expression of key enzymes involving in D-lactic acid production were conducted for selection of the novel D-lactic acid isolate with the high yield, productivity, and optical purity. From the screening experiment, the novel isolate, NK26-11T, was obtained. The isolate NK26-11T was a Gram-stain-positive, catalase-positive, facultatively anaerobic, spore-forming, rod-shaped bacterium isolated from soil sample in Thailand. This isolate homofermentatively fermented glucose for D-lactic acid and grew at the wide range of temperature between 20 and 45 °C and the pH of 5-8.5. The cell-wall peptidoglycan of NK26-11T contained meso-diaminopimelic acid. The major respiratory quinone was menaquinone 7 (MK-7), the DNA G+C content was 42.6 mol%, and the major cellular fatty acids were anteiso-C15:0 and anteiso-C17:0. On the basis of 16S rRNA gene sequences analysis, the isolate NK26-11T was closely related to Bacillus solimangrovi JCM 18994T (93.89% 16S rRNA gene sequence similarity), Pullulanibacillus naganoensis LMG 12887T (93.32%), Sporolactobacillus inulinus NRIC 1133T (92.99%), Tuberibacillus calidus JCM 13397T (92.98%) and Thalassobacillus devorans DSM 16966T (˂90.93%). The isolate NK26-11T represents a novel species of a new genus between Bacillus and Sporolactobacillus clusters, for which the name Terrilactibacillus laevilacticus gen. nov., sp. nov. was proposed. The type strain of the type species is NK26-11T (=LMG 27803T =TISTR 2241T =PCU 335T). From the fermentation screening of the selected D-lactic acid producing isolates, it was found that Terrilactibacillus laevilacticus SK5-6 exhibited a good D-lactate production performance (99.27 g/L final lactate titer with 0.90 g/g yield, 1.38 g/L.h, and 99.00% D-enantiomer equivalent) compared with other Sporolactobacillus sp. and Terrilactibabacillus sp. This isolate could ferment a wide range of sugars for D-lactic acid. Unlike the typical D-lactic acid producers, such as catalase negative Sporolactobacillus sp., T. laevilacticus SK5-6 acquired catalase activity; therefore, a 2-phase fermentation was simply employed for D-lactic acid production. Under an aerobic preculture stage, high-cell-density biomass was rapidly obtained as the result of aerobic respiration. At the correct physiological stage (inoculum age) and a proper concentration of biomass (inoculum size) transferred to the fermentation stage, SK5-6 rapidly converted glucose into D-lactic acid under anaerobic conditions resulting in a high final lactic acid titer (102.22 g/L), yield (0.84 g/g), and productivity (2.13 g/L.h) without byproduct formation. It was found that SK5-6 exhibited both fermentation kinetic and expression level of the key enzymes higher than those of S. laevilacticus, a catalase negative D-lactate producer. Low phosphofructokinase activity revealed that glycolysis controlled the apparent D-lactic acid productivity by SK5-6. Increasing the pH during fermentation phase activated the activity of D-LDH (D-lactate dehydrogenase) beyond that of L-LDH, resulting in the high optical purity of D-lactate, while the acidic pH promoted the activities of the kinases in glycolysis. The conversion of L-lactate to D-lactate by isomerase was also observed during fermentation. The findings in this study demonstrated the remarkable characteristics of SK5-6, in particular, a high product yield was obtained without byproduct formation. From the key characteristics of SK5-6, this isolate can be claimed as an industrial D-lactic acid producer.