Marisia - Maros Megyei Múzeum Évkönyve 35/2. (2015)
Zoology
S. NEAGU, R. COJOC, M. ENACHE, I. GOMOIU, G. GHEMES, A. GHEORGHE, M. TUDORACHE al., 2004) and Klebsiella pneumoniae shows the ability to transform glycerol in 3-hydroxypropionic acid (Wang et ah, 2012; Li et al., 2013). On the other hand, Escherichia coli strains convert glycerol to hydrogen (Dharmadi et al., 2006; Sharninghausen et al., 2014) and other value added products like ethanol, succinic, acetic or lactic acids and fatty acids omega—3 polyunsaturated (Yang et al., 2012). The present paper deals with the growth of moderately halophilic microorganisms isolated from saline environments in Romania on culture media supplemented with various concentrations of waste glycerol. The resulted biomass and supernatant culture were tested as biocatalyst in the bio transformation processes of waste glycerol into value-added products (e.g. glycidol and glycerol carbonate). Materials and methods Culture and media The investigated halophilic microorganisms were isolated from saline lake Balta Alba, located in Romania in Buzau-Braila counties, aboutl70 km East of Bucharest. For the isolation of strains, one milliter of water sample was mixed in a Petri dish with molten agar medium (MH) containing (g/1): NaCl - 100, MgCl2 x 6H20 - 7, MgS04 x 7H,0 - 9.6, CaCl2 x 2H20 — 0.36, KC1 — 2, NaHCO, - 0.06, NaBr - 0.026, glucose - 1, proteose peptone — 5, yeast extract - 10 (Ventosa et al.,1989). The medium pH was adjusted to 7.0 — 7.2 before autoclaving. After solidification, the plates were incubated at 28°C for 7—10 days and after this period the colony forming units (c.f.u.) number was counted. In further experiments the culture medium were used in liquid form for growth of both wild investigated halophilic strains and Marinococcus halophilus strain JCM 2472 (courtesy of dr. Takashi Itoh — Japan Collection of Microorganisms). Summary characterization of bacterial cells The preliminary characterization of the tested strains in order to assign them to the group of moderately halophilic bacteria followed the growth in the presence of bile salts and chloramphenicol and the range of NaCl concentrations for growth. In this way, the culture media were supplemented with 0,002% chloramphenicol or 0,004% sodium deoxycholate. The strains able to grow in the presence of sodium deoxycholate and unable to grow in the presence of chloramphenicol were recorded as bacteria. In order to estimate the range of NaCl and optimum concentration of NaCl for growth, the content of NaCl from culture media was varied from zero until to 5M. The growth of the investigated strains was recorded on solidified media. Type of waste glycerol The ability of investigated strains to use various types of waste glycerol was tested by using culture media supplemented with 1% glycerol. The investigated types of glycerol were represented by manufacturer suppliers: Medias source, Slobozia source, sun flower oil Medias, rapeseed oil and palm oil. In some experiments the NaCl content in culture media was adjusted to 5% or 10%. The growth of the strains was recorded as optical density at 660 nm by using UV-VIS spectrophotometer (Analytik Jena). Biotransformation of glycerol into glycerol carbonate and glycidol The reaction mixture contained glycerol and dimethyl carbonate (1:10 molar ratio) mixed with 100 mg cell suspension or 100 pL supernatant. After 24 h (reaction time), the biocatalyst was separated from the reaction mixture by centrifugation. The liquid phase (supernatant) was dried under vacuum conditions and re-suspended in 200 pL derivatization reagent (BTFA: pyridine= 1:1, v/v). GC-FID analysis was performed in order to determine the quantitative composition in glycidol and glycerol carbonate. 68
