Angelova R. , Iliev M., Mitova M., Groudeva V.

1. OPTIMIZATION OF THE CULTIVATION OF NEUTROPHILIC IRON BACTERIA FROM THE GROUP SPHAEROTILUS - LEPTOTHRIX Angelova R., Iliev M., Mitova M., Groudeva V. Department of General and Industrial Microbiology,Faculty of Biology, Sofia University, Sofia 1421, Bulgaria Introduction The bacteria of the group Sphaerotilus - Leptothrixare typical sheathed ß- Proteobacteria, with variation of their morphology and physiology and able to oxidize Fe2+ at neutral pH. As a result of this oxidation insoluble ferric hydroxides witch are of great interest for different nanobiotechnologies (as pigments, adsorbents, catalysts, ferrofluids etc. [1, 2]) are formed. Aim The aim of this investigation is connected with optimization of the conditions for cultivation of these bacteria under laboratory conditions. The bacteria are isolated from natural habitats (Vitosha Mountain, Bulgaria) and identified as members of the group Sphaerotilus-Leptothrix by the methods of classical and molecular taxonomy. Materials and Methods Sampling region Results and discussion Six different elective nutrient media with variation of the Fe source as well as different methods for cultivation were used. The oxidation of Fe2+, changes of the pH, changes of the morphology of the cells and the type of the ferric hydroxides formed were tested during the all period of the cultivation. The culture media used are suitable for obtaining enriched cultures and isolating pure cultures of the genus Leptothrix. This was confirmed by classical taxonomy and by molecular methods. The Fehrenbach flasks provide better conditions for the target bacteria with numbers greater than in Roux flasks and the installation for aerobic cultivation. The typical morphology of the cells was confirmed by light and electron microscopy. Fig. 1 shows cells with shape typical for this genus. Fig. 2 shows the amplification profile mofA - PCR. Analysis of samples from the area of sampling for the presence of iron bacteria carried out by light and electron microscopy showing the expected iron bacteria morphology and the typical sheaths they create. a b Typical deposits of iron bacteria in Vitosha Mountain (1783 m altitude) c d Types of cultivation vessels: Fig.1. (а, b) SEM of bacteria of the genus Leptothrix cultured inmedium: a) Panoramicview;с) Typical cell shape with a rough surface; (c) SEM of bacteria cultured in an Isolation medium RouxFlasks Molecular methods(PCR detection assay) 5 μm Microscopic images of the samples from the area of ​​sampling: (a) and (b) SEM of bacterial cells, (с) SEM of sheaths – 10 000x, (d) light microscopy image of sheaths. Culture media used for multiplication and selective cultivation of iron bacteria FehrenbachFlasks Fig. 2. Amplification profile of mofA-PCR The best results were obtained by cultivation in Lieske and Adler media in which FeSO4 were used as a source of Fe2+. The optimal growing conditions are at temperature of about 16 - 20 °C, a pH about 7.0 and each change of the pH values below 5.0 and up to 8.0 strongly inhibited the bacterial growth. The type of the ferric hydroxides formed strongly depended on the composition of the media used. The cultivation under static conditions was much more efficient for sheaths formation in comparison with the cultivation under dynamic conditions. In any case the formation of the sheaths was very slowly and such structures were formed after at least 20 days cultivation. The reason for not forming the seats under laboratory conditions is not clear up to now. Installation for aerobiccultivation Isolation and storage of pure cultures: pure cultures were prepared on an isolation medium (IM) through a standard procedure using swabs from bacteria developedin culture vessels. The cultures were isolated from single colonies on agar slanting strains stored on the same nutrient media. Conclusions The cultivation of the bacteria from the group Sphaerotilus-Leptothrix in laboratory conditions is connected with many difficulties and slow growth without formation of the sheath structures. Additional investigations must be done for understanding of this phenomenon. Identification of isolates by classical taxonomy. A taxonomic scheme was used according to the identification keyof Bergey (Bergey’s manual of determinative bacteriology, 8th ed, 1989), involving morphological and biochemical characteristics. Molecular methods(PCR detection assay) The bacterial cells were harvested by centrifugation (4 500rpm/10 min), the cell pellet was washed with PBS and subjected to DNA isolation with PrepMiniSpinKit (GEHealthcare). The published sequence of mofA gene (GenBank № Z25774.3) was chosen as a specific target for PCR detection of Leptothrix spp. The specific primers were constructed with Primer-Blast Software. F1_ thrixis5’-TGT-TCG-AGC-CGG-TGT-TCG-GC-3’, and R1_ thrix5’-GAA-TCG-ATC-GCG-ACC-GGC-GT-3’. The PCR mixture contained 1 µМof each primer (SenseandAntisense), 0,2 mМdNTPs, Taqbuffer 1х (Invitrogen), 1,5 mМMgCl2, 2,5 UTaqpolymeraseand 5 µl (10-100 ng) totalDNA (Ready-To-GoPCRkit (GEHealthcare).The PCR programme consisted of an initial denaturating step 95ºС/5min, followed by 35 cycles (95ºС/1min; 54ºС/1min; 72ºС/1min) and a final extension step at 72ºС for 5 min. All reactions were carried out on an EppendorfThermocycler (Eppendorf). References: [1] M. Sawayama et al., Isolation of a Leptothrix Strain, OUMS1, from Ocherous Deposits in Groundwater, Curr. Microbiol. 63 (2011) 173–180. [2] H. Hashimoto et al., Synthesis and magnetic properties of magnetite-silicate nanocomposites derived from iron oxide of bacterial origin, Mater. Chem. Phys. 136 (2012) 1156 -1161. This work was supported by the Bulgarian National Science Fund under project DID 02/38/2009