The ability of Clostridium bifermentans strains to lactic acid biosynthesis in various environmental conditions

Katarzyna Leja 0 Kamila Myszka 0 Katarzyna Czaczyk 0 0 Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences , Wojska Polskiego 48, 60-627, Poznan, Poland Clostridium bifermentans strains, isolated from a manure, were examinated for their ability to produce lactic acid from PY medium with glycerol under different pH conditions and when PY medium was supplemented with saccharides such as fructose, sorbitol, glucose, mannose, mannitol, maltose, xylose, raffinose, and arabinose. In the last test performed, the ability of investigated strains to produce lactic acid from mixed carbon source (glycerol plus saccharide) was checked. The strains of Cl. bifermentans, designated as CB 371, CB 374, and CB 376 grew and produced lactic acid on PY medium irrespective of pH and the carbon source used. The optimal lactic acid production on PY medium with glycerol was obtained at pH of 7.0 in case of CB 371 and 376 (19.63 g/L and 16.65 g/L, accordingly) and at pH 8.0 in case of CB 374 (13.88 g/L). The best productivity of lactic acid on PY media by CB 371, CB 374, and CB 376 (above 30 g/L) was observed when mannitol was used as a carbon source. The mixed carbon source did not increase productivity of lactic acid by Cl. bifermentans. The yield of lactic acid was approximately equal to the yield of lactic acid obtained on the medium with only glycerol and lower than in medium with only mannitol. Thus, from the environmental point of view it is more beneficial to use the medium with waste-type material only, such as glycerol. - Introduction Cl. bifermentans was first isolated by Tissier and Martelly in 1902. A taxonomic relationship to Cl. sordelli, isolated first in 1922, resulted in the symptomatic fact that both strains were identified as one species (Brooks & Epps 1958). As late as in 1963, Cl. bifermentans and Cl. sordelli were distinguished as separate species of the genus Clostridium. As a main factor whose influence was taken into consideration here was pathogenicity: Cl. sordelli was described as a pathogenic variant of non-pathogenic Cl. bifermentans. Additionally, these two bacterial species can be distinguished one from another in the ureaseproduction test. By 1955, the idea of separating Cl. bifermentans and Cl. sordelli gained acceptance of researchers. The original isolate of Clostridium bifermentans was named Bacillus bifermentans sporogenes (Clark and Hall 1937), and later re-named B. bifermentans (Bergey et al. 1923), in accordance with the principle of binominal nomenclature (Brooks & Epps 1958). The main sources of Cl. bifermentans occur in water, soil, sewage (Nachman et al. 1989), sludge, and animal faeces (Wang et al. 2003). Cl. bifermentans is able to produce a wide range of metabolites such as acetic, butyric and formic acids (Wu & Yang 2003), ethanol, butanol, aceton (Khanal et al. 2004), carbon dioxide, hydrogen, and nitrogen (Levin et al. 2006). However, the metabolic pathway of Cl. bifermentans has not been investigated in detail so far. The aim of this work thus was to investigate the possibility of lactic acid production by Cl. bifermentans when, as carbon source, glycerol or other saccharides are added to the cultivation medium as well as under low- and high-pH stress on glycerol medium. Materials and methods Source of strains Cl. bifermentans strains (KM 371, KM 374 and KM 376) were isolated from samples that were collected from a manure in the Wielkopolska Region, Poland. Samples were collected in sterile plastic jars and stored in refrigerator until experimentations. Liquid samples were then inoculated to the modified PY medium according to Biebl and Sper (2002). The isolating process is described in more detail in Myszka et al. (2012). Cultivation medium The modified PY medium consisted of (g/L): BactoPeptone 10; yeast extract 10; CaCl2, MgSO4 7H2O 0.96; K2HPO4 2; NaHCO3 20; NaCl 4 was used. As a source of carbon in the PY medium, glycerol (50 g/L) or saccharides such as fructose, sorbitol, glucose, mannose, mannitol, maltose, xylose, raffinose, and arabinose (50 g/L) (Sigma-Aldrich) were added. The pH of the PY medium without regulation is of the value of 8.6 using 10% solution of NaOH and HCl (Sigma Aldrich). Batch fermentation A preculture was carried out in a 500 ml flask containing 300 ml PY medium with glycerol at 37C for 24 h. It was inoculated into a 5 L bioreactor (Sartorius Stedim, Germany) with 3 L PY medium (with glycerol or, respectively, saccharide). According to Myszka et al. (2012), a blanket of a high-purity grade gas mixture of 5% O2 and 95% CO2 was maintained through 24 h. Gas flow rate was at up to 1.0 L/min only. During the first 24 h of cultivation the level of 5% of oxygen was automatically maintained (the stirrer speed varied between 200 and 500 rpm). After 24 h of the duration of the process the stirrer speed was regulated to a constant value of 200 rpm. The fermentation was run at 30C for 7 days. Fermentation at various pH (...truncated)