Screening and characterization of succinic acid producing bacteria and its production optimization

Abstract

Succinic acid producing bacteria were isolated from 6 sources in Thailand. Firstly, a total 310 isolates, only 51 isolates were screened for their succinic acid production by selective medium plate. They showed positive on TLC method and they were confirmed to have succinic acid producing ability using HPLC. Succinic acid was obtained in the range of 0.553-52.028 g/l. They were divided into 9 groups based on morphological, physiological and biochemical characteristics. Isolates produced high succinic acid from each group were selected to study 16S rRNA gene sequence. Isolate CN1-OB13 (Group I) was closely related to Escherichia fergusonii ATCC 35469T (99.87%). Isolate PCH6-3 (Group II) was closely related to Lactobacillus reuteri JCM 1112T (99.71%). Isolate AY5-bA2 (Group III) was closely related to Lactobacillus ruminis NBRC 102161T (99.71%). Isolate AY5-bB4 (Group IV) was closely related to Clostridium sporogenes DSM 795T (99.78%). Isolate PCH2-1 (Group V) was closely related to Enterococcus faecium CGMCC 1.2136T (99.86%). Isolate NP1-A2 (Group VI) was closely related to Enterococcus faecalis ATCC 19433T (99.86%). Isolate CN2-OA2 (Group VII) was closely related to Enterococcus avium ATCC 14025T (100%). Isolate NS15-bA2 (Group VIII) was closely related to Enterococcus hirae ATCC 9790T (100%). Lastly, isolate NS15-dA1 (Group IX) was closely related to Enterococcus durans CECT411T (99.89%). Isolate NS15-bA2 and NS15-dA1 could produce the highest succinic acid of 52.028 and 50.862 g/l from 60g/l of glucose, respectively and they were no reported of succinic acid production from other research. Thus these two isolates were selected to study optimization of succinic acid production. Next, the medium composition that affected to the succinic acid production; carbon sources (glucose concentration), nitrogen sources (organic and inorganic), pH and temperature were investigated. The optimum conditions on succinic acid production by isolate NS15-dA1 and NS15-bA2 was 60 g/l of glucose as a carbon source, 30 g/l of yeast extract (for isolate NS15-dA1) and 30 g/l of tryptone (for isolate NS15-bA2) as a nitrogen source, 0.2 g/l of CaCl₂.2H₂O, 0.2 g/l of MgCl₂.6H₂O, 0.07 g/l of MnCl₂, 4.4 g/l of Na2HPO₄, 3.3 g/l of NaH₂PO₄, 30 g/l of MgCO₃ at pH 7.0 and 37°C for 24 h. The highest succinic acid of 51.692±0.1707 g/l and 53.051±0.3538 g/l were obtained, respectively.